Temozolomide compounds, polymers prepared therefrom, and method of treating a disease

ABSTRACT

A temozolomide compound according to formula (I)is described, wherein R1, L1, and X are defined herein. The temozolomide compound can be used to prepare polymers comprising temozolomide. Additionally, the polymers comprising temozolomide can be particularly useful in the treatment of certain diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuing application of U.S. application Ser.No. 16/735,910, filed Jan. 7, 2020, which is a continuing application ofU.S. application Ser. No. 15/709,862, filed Sep. 20, 2017, which claimsthe benefit of U.S. Provisional Application No. 62/398,078, filed Sep.22, 2016, each of which are incorporated by reference herein in theirentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH & DEVELOPMENT

This invention was made with government support under award numberDMR-0820506 awarded by the National Science Foundation MaterialsResearch Science & Engineering Center (MRSEC) on Polymers and underaward number R21 CA167674 awarded by the National Institutes of Health.The government has certain rights in the invention.

BACKGROUND

Temozolomide (TMZ) is a first-line chemotherapeutic indicated fortreating patients diagnosed with glioblastoma. Administered orally, TMZis a lipophilic, acid-stable DNA alkylating agent that efficientlycrosses the blood-brain barrier and presents mild side effects. See,e.g., Adamson, C.; Kanu, O. O.; Mehta, A. I.; Di, C.; Lin, N.; Mattox,A. K.; Bigner, D. D. Glioblastoma Multiforme: A Review of Where We HaveBeen and Where We Are Going. Expert Opin. Investig. Drugs 2009, 18,1061-1083. Serving as a small molecule prodrug, TMZ hydrolyzesspontaneously at physiologic pH liberating methyldiazonium cations thatelicit antitumor activity by methylation of guanine and adeninenucleobases. See, e.g., Zhang, J.; Stevens, M. F. G.; Bradshaw, T. D.Temozolomide: Mechanisms of Action, Repair and Resistance. Curr. Mol.Pharmacol. 2012, 5, 102-114; Newlands, E. S.; Stevens, M. F. G.; Wedge,S. R.; Wheelhouse, R. T.; Brock, C. Temozolomide: A Review of ItsDiscovery, Chemical Properties, Pre-Clinical Development and ClinicalTrials. Cancer Treat. Rev. 1997, 23, 35-61. Owing to the slightlyalkaline microenvironment of brain tumors (see, e.g., Rottenberg, D. A.;Ginos, J. Z.; Kearfoot, K. J.; Junck, L.; Bigner, D. D. In VivoMeasurement of Regional Brain Tissue pH Using Positron EmissionTomography. Ann. Neurol. 1984, 15, S98-102) this pH-induced mechanism ofaction confers selective cytotoxicity for malignant versus healthy braintissue (see, e.g., Zhang, J.; Stevens, M. F. G.; Bradshaw, T. D.Temozolomide: Mechanisms of Action, Repair and Resistance. Curr. Mol.Pharmacol. 2012, 5, 102-114) resulting in limited off-target toxicity.Unfortunately, in vivo decomposition and clearance of TMZ is rapid, andfrequent dosing is required to retain antitumor activity. See, e.g.,Stevens, M. F. G.; Hickman, J. A.; Langdon, S. P.; Chubb, D.; Vickers,L.; Stone, R.; Baig, G.; Goddard, C.; Gibson, N. W.; Slack, J. A.;Newton, C.; Lunt, E.; Fizames, C.; Lavelle, F. AntitumorImidazo[5,1-d]-1,2,3,5-Tetrazin-4(3H)-One (CCRG 81045: M & B39831), aNovel Drug with Potential as an Alternative to Dacarbazine. Cancer Res.1987, 47, 5846-5852. Moreover, efficacy is often hampered bychemoresistance primarily induced by O⁶-methylguanine-DNAmethyltransferase (MGMT), a native enzyme that repairs damaged DNAfollowing methylation. See, e.g., Newlands, E. S.; Stevens, M. F. G.;Wedge, S. R.; Wheelhouse, R. T.; Brock, C. Temozolomide: A Review of ItsDiscovery, Chemical Properties, Pre-Clinical Development and ClinicalTrials. Cancer Treat. Rev. 1997, 23, 35-61. Clinical examination hasshown that MGMT activity in glioblastoma tumors can be depleted bysustained TMZ exposure (see, e.g., Tolcher, A. W.; Gerson, S. L.; Denis,L.; Geyer, C.; Hammond, L. A.; Patnaik, A.; Goetz, A. D.; Schwartz, G.;Edwards, T.; Reyderman, L.; Statkevich, P.; Cutler, D. L.; Rowinsky, E.K. Marked Inactivation of O⁶-Alkylguanine-DNA Alkyltransferase Activitywith Protracted Temozolomide Schedules. Br. J. Cancer 2003, 88,1004-1011), and that recurrent glioblastoma treated with protracteddosing schedules can improve tumor response. See, e.g., Wick, W.;Steinbach, J. P.; Kuker, W. M.; Dichgan, J.; Bamberg, M.; Weller, M. Oneweek On/One Week Off: a Novel Active Regimen of Temozolomide forRecurrent Glioblastoma. Neurology 2004, 62, 2113-2115; Jauch, T.; Hau,P.; Bogdahn, U. Re-challenge with Temozolomide (TMZ) at Recurrence inHigh-Grade Gliomas (HGG). J. Clin. Onocol. 2007, 25, 2034; Wick, A.;Felsberg, J.; Steinbach, J. P.; Herrlinger, U.; Platten, M.; Blaschke,B.; Meyermann, R.; Reifenberger, G.; Weller, M.; Wick, W. Efficacy andTolerability of Temozolomide in an Alternating Weekly Regimen inPatients with Recurrent Glioma. J. Clin. Oncol. 2007, 25, 3357-3361;Berrocal, A.; Perez Segura, P.; Gil, M.; Balana, C.; Garcia Lopez, J.;Yaya, R.; Rodriguez, J.; Reynes, G.; Gallego, O.; Iglesias, L.; GENOMCooperative Group. Extended-Schedule Dose-Dense Temozolomide inRefractory Gliomas. J. Neurooncol. 2010, 96, 417-422. As thesealternative regimens can increase occurrence of dose-limitinghematoxicity, an alternative therapeutic modality that extends TMZcirculation and masks drug-associated toxicity is desired.

Polymer-drug conjugation is effective for augmenting the antitumorefficacy of many small molecule chemotherapeutics, affordingwater-soluble prodrugs with improved pharmacokinetic behavior. See,e.g., Larson, N.; Ghandehari, H. Polymeric Conjugates for Drug Delivery.Chem. Mater. 2012, 24, 840-853; Fox, M. E.; Szoka, F. C.; Frdchet, J. M.J. Soluble Polymer Carriers for the Treatment of Cancer: The Importanceof Molecular Architecture. Acc. Chem. Res. 2009, 42, 1141-1151. As aresult of the leaky vasculature and poor lymphatic drainage of solidtumors, polymer therapeutic accumulation in malignant tissue isenhanced, leading to potentiated antitumor activity and reducedoff-target toxicity. See, e.g., Maeda, H.; Nakamura, H.; Fang, J. TheEPR Effect for Macromolecular Drug Delivery to Solid Tumors: Improvementof Tumor Uptake, Lowering of Systemic Toxicity, and Distinct TumorImaging In Vivo. Adv. Drug Delivery Rev. 2013, 65, 71-79.

Various delivery strategies have been explored for improving TMZtherapy, including encapsulation in nanoparticles and liposomes forsystemic administration, as well as entrapment in hydrogels, degradablematrices, and microspheres for localized treatments (see, e.g., Fang,C.; Wang, K.; Stephen, Z. R.; Mu, Q.; Kievit, F. M.; Chiu, D. T.; Press,O. W.; Zhang, M. Temozolomide Nanoparticles for Targeted GlioblastomaTherapy. ACS Appl. Mater. Interfaces 2015, 7, 6674-6682; Kim, S-S.;Rait, A.; Kim, E.; DeMarco, J.; Pirollo, K. F.; Chang, E. H.Encapsulation of Temozolomide in a Tumor-Targeting Nanocomplex EnhancesAnti-Cancer Efficacy and Reduces Toxicity in a Mouse Model ofGlioblastoma. Cancer Lett. 2015, 369, 250-258; Dong, J.; Zhou, G.; Tang,D.; Chen, Y.; Cui, B.; Dai, X.; Zhang, J.; Lan, Q.; Huang, Q. LocalDelivery of Slow-Releasing Temozolomide Microspheres InhibitsIntracranial Xenograft Glioma Growth. J. Cancer Res. Clin. Oncol. 2012,138, 2079-2084; Fourniols, T.; Randolph, L. D.; Staub, A.; Vanvarenberg,K.; Leprince, J. G.; Prdat, V.; des Rieux, A.; Danhier, F.Temozolomide-Loaded Photopolymerizable PEG-DMA-Based Hydrogel for theTreatment of Glioblastoma. J. Controlled Release 2015, 210, 95-104). Todate, only one example of a polymer-TMZ therapeutic, prepared usingmetabolically-derived poly(D-L-malic acid), has been reported. See,e.g., Patil, R.; Portilla-Arias, J.; Ding, H.; Inoue, S.; Konda, B.; Hu,J.; Wawrowsky, K. A.; Shin, P. K.; Black, K. L.; Holler, E.; Ljubimova,J. Y. Temozolomide Delivery to Tumor Cells by a Multifunctional NanoVehicle Based on Poly(β-t-malic acid). Pharm. Res. 2010, 27, 2317-2329.While this conjugate demonstrated in vitro activity against glioblastomatumor cells, including a naturally chemoresistant cell line, chemicaland physical characterization of the polymer prodrug was limited, andthe synthetic strategy employed was not suitable for preparingwell-defined structures with tunable drug content.

Glioblastoma is a cerebral neoplasm that represents the most prolificmalignant central nervous system tumor diagnosed in the United States,with nearly 11,000 new cases presented annually. See, e.g., Ostrom, Q.T.; Gittleman, H.; Fulop, J.; Liu, M.; Blanda, R.; Kromer, C.; Wolinsky,Y.; Kruchko, C.; Barnholtz-Sloan, J. S. CBTRUS Statistical Report: Brainand Central Nervous System Tumors Diagnosed in the United States in2008-2012. Neuro Oncol. 2015, 17, iv1-iv62. Characterized by substantialhistologic and genetic heterogeneity, glioblastoma is an infiltrativeastrocytoma with dismal patient prognosis. Even with early detection,median survival of only 12 to 15 months is expected following treatment.See, e.g., Omura, A.; DeAngelis, L. M. Glioblastoma and Other MalignantGliomas. JAMA 2013, 310, 1842-1850; Wen, P. Y.; Kesari, S. MalignantGliomas in Adults. N. Engl. J. Med. 2008, 359, 492-507. Standard of carefor patients with newly diagnosed glioblastoma includes surgicaldebulking to resect maximal solid tumor tissue, followed bychemoradiotherapy and adjuvant chemotherapy with TMZ. Though therapeuticintervention can temporarily arrest disease progression, recurrence isgenerally inevitable.

Accordingly, there remains a continuing need for improved deliverysystems for TMZ (e.g., polymer delivery systems), particularly for thetreatment of cancer (e.g., glioblastoma).

BRIEF SUMMARY

One embodiment is a temozolomide compound of structure (I)

wherein X is —O— or —NR^(a)—, wherein Ra is hydrogen or a C₁₋₆ alkylgroup; L¹ is a divalent C₁₋₁₂ alkylene group, di(C₂₋₁₂) alkylenedisulfide group, C₂₋₁₂ alkylene ester group, C₆₋₂₀ arylene group, C₁₋₂₀alkylene oxide group, or C₁₋₁₂ alkylene sulfide group; n is 0 or 1; andR¹ is a group of the formula H₂C═C(R^(b))—(C═O)—W—, wherein R^(b) ismethyl, hydrogen, fluoro, cyano, or trifluoromethyl, and W is —O— or—NH—; an alkenyl group; an alkynyl group; an aldehyde group; a ketonegroup; a thiol group; a pentafluorophenyl group, a pyridyl disulfidegroup, a zwitterionic group, a glutathione group, a thiamine group, or apoly(ethylene glycol) group.

Another embodiment is a polymer comprising repeating units comprisingtemozolomide derived from the temozolomide compound, and optionally,repeating units of formula (II), formula (III), or a combination thereof

wherein in each occurrence of the repeating units of formula (II) R² isa hydrogen or a C₁₋₆ alkyl group; V is —O— or —NH—; and R³ is azwitterionic group, a poly(C1-6 alkylene oxide) group, ahydroxy-substituted C₁₋₆ alkyl group, or a C₁₋₁₂ alkyl group; andwherein in each occurrence of the repeating units of formula (III) R² isa hydrogen or a C₁₋₆ alkyl group; and R⁶ is a C₆₋₂₀ aryl group.

Another embodiment is a poly(ethylene glycol)-temozolomide conjugatecomprising a poly(ethylene glycol) having at least two chain endsconjugated to a temozolomide compound.

Another embodiment is a method of treating a disease, the methodcomprising administering a therapeutically effective amount of acomposition comprising the polymer.

These and other embodiments are described in detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Figures are of exemplary embodiments:

FIG. 1 is a chemical scheme depicting the synthesis of TMZ-methacrylate(shown as compound 2).

FIG. 2 shows the UV/Vis absorption spectra of TMZ and compound 2 intrifluoroethanol at concentrations of 0.01 milligrams per milliliter(mg/ml).

FIG. 3 shows the UV-Vis absorption spectra of TMZ in 1:1methanol:dimethylsulfoxide (MeOH:DMSO) at 0.01 mg/ml. Solutions werestirred at room temperature, 50° C., or 70° C. and analyzed atdesignated times.

FIG. 4 shows the UV-Vis absorption spectra of TMZ in DMSO at 0.01 mg/ml.Solutions were stirred at room temperature, 50° C., or 70° C. andanalyzed at designated times.

FIG. 5 shows the UV-Vis absorption spectra of TMZ in acetonitrile (ACN)at 0.01 mg/ml. Solutions were stirred at room temperature, 50° C., or70° C. and analyzed at designated times.

FIG. 6 shows the UV-Vis absorption spectra of TMZ in trifluoroethanol(TFE) at 0.01 mg/ml. Solutions were stirred at room temperature, 50° C.,or 70° C. and analyzed at designated times.

FIG. 7 is a chemical scheme depicting the copolymerization ofmethacryloyloxyethyl phosphorylcholine (MPC) and TMZ-methacrylate.

FIG. 8 shows the UV-Vis absorption spectra of polyMPC-TMZ copolymers inTFE.

FIG. 9 shows the ¹H-NMR (500 MHz) spectrum of polyMPC-TMZ (3D) obtainedin deuterated trifluoroethanol (TFE-d₃).

FIG. 10 shows the gel permeation chromatograms of polymers 3A-3D,obtained by gel permeation chromatography (GPC) eluting with TFE.

FIG. 11 shows TFE GPC chromatograms of compound 2 and polymer 3A withdetection by UV-Vis absorbance at k=323 nm. For copolymer 3A, a singlepolymer fraction was observed (elution time of 26.6 minutes), with anotable absence of residual compound 2 (elution time of 34.2 minutes).The chromatogram of 3A is representative of copolymers 3B-3D.

FIG. 12 shows cell viability of U87MG cells in the presence of differentPolyMPC-TMZ conjugates (random co-polymers).

FIG. 13 shows cell viability of U87MG cells in the presence of differentPolyMPC-TMZ conjugates (block co-polymers).

FIG. 14 is a schematic illustration showing the encapsulation of atherapeutic in a polyMPC-TMZ nanoparticle.

FIG. 15 shows UV-Vis spectra of a polyMPC-TMZ block copolymer, pristineTM), and TMZ-loaded polyMPC-TMZ NPs. The increase in absorbance for theTMZ-characteristic absorption (λ=328-336 nm) confirms TMZ encapsulationin the nanoparticles.

DETAILED DESCRIPTION

The present inventors have discovered new synthetic compositions oftemozolomide (TMZ). Through the use of new functional derivatives ofTMZ, homo- and copolymers comprising TMZ can be prepared, including bothwater-soluble TMZ-containing polymers, as well as water-insolubleformulations. The TMZ-containing polymers described herein are thoughtto be particularly useful in the treatment of certain cancers. TheTMZ-containing polymers described herein can advantageously be preparedas injectable TMZ-containing formulations for systemic treatment of suchdiseases, or as implantable TMZ-containing formulations. Without wishingto be bound by theory, it is believed that the increased size of theTMZ-containing polymers relative to TMZ itself can extend the in vivocirculation time, reduce treatment frequency, and provide opportunitiesfor increased drug accumulation in tumors through the enhancedpermeability and retention effect (e.g., when used as an injectableformulation). Additionally, the TMZ-containing polymers described hereincan potentially assemble in solution resulting from hydrophobic druginteractions, which can serve to enhance TMZ stability and lifetime inthe bloodstream. Small molecule, water-soluble TMZ derivatives are alsodiscussed.

Accordingly, an aspect of the present disclosure is a temozolomidecompound. In some embodiments, the temozolomide compounds of the presentdisclosure advantageously include a functional group that can be usedfor subsequent conjugation of the temozolomide compound to, for example,a polymer carrier, or other drug delivery platform. In otherembodiments, the temozolomide compounds of the present disclosure caninclude a water-solublizing group which can provide temozolomide as asmall molecule (i.e., not conjugated to a polymer scaffold) in a watersoluble form. In particular, the temozolomide compound has the structureaccording to formula (I)

wherein X is —O— or —NR^(a)—, wherein Ra is hydrogen or a C₁₋₆ alkylgroup. In some embodiments, X is —O—. In some embodiments, X is—NR^(a)—, wherein Ra is preferably hydrogen (i.e., X is —NH—). L¹ is adivalent C₁₋₁₂ alkylene group, di(C₂₋₁₂ alkylene) disulfide group (e.g.,—(C₂₋₁₂ alkylene)-S—S—(C₂₋₁₂ alkylene)-), C₂₋₁₂ alkylene ester group,C₆₋₂₀ arylene group, C₁₋₂₀ alkylene oxide group, or C₁₋₁₂ alkylenesulfide group. In some embodiments, L¹ can be a divalent C₁₋₁₂ alkylenegroup, di(C₂₋₁₂ alkylene) disulfide group (e.g., —(C₂₋₁₂alkylene)-S—S—(C₂₋₁₂ alkylene)-), C₂₋₁₂ alkylene ester group, C₆₋₂₀arylene group, or C₁₋₂₀ alkylene oxide group. In some embodiments, L¹ ispreferably a divalent C₁₋₁₂ alkylene group, di(C₂₋₁₂ alkylene) disulfidegroup, or C₁₋₂₀ alkylene oxide group, more preferably a divalent C₁₋₁₂alkylene group or di(C₂₋₁₂ alkylene) disulfide group, even morepreferably a divalent C₁₋₁₂ alkylene group. In some embodiments, L¹ is aC₁₋₆ alkylene group. In some embodiments, L¹ is a C₂₋₆ alkylene group.Further in formula (I), n can be 0 or 1.

R¹ in formula (I) is a group of the formula H₂C═C(R^(b))—(C═O)—W—,wherein R^(b) is methyl, hydrogen, fluoro, cyano, or trifluoromethyl,and W is —O— or —NH—; an alkenyl group; an alkynyl group; an aldehydegroup; a ketone group, or a thiol group. In some embodiments, R¹ is agroup of the formula H₂C═C(R^(b))—(C═O)—O—, wherein R^(b) is methyl,hydrogen, fluoro, cyano, or trifluoromethyl. In some embodiments, R^(b)is a hydrogen, and R1 is an acrylate group. In some embodiment, R^(b) isa methyl group, and R¹ is a methacrylate group. In some embodiments, R¹is a group of the formula H₂C═C(R^(b))—(C═O)—NH—, where R^(b) ispreferably a methyl group, and R¹ is a methacrylamide group. In someembodiments, R¹ is a group of the formula H₂C═C(R^(b))—(C═O)—NH—, whereR^(b) is preferably a hydrogen, and R¹ is an acrylamide group. In someembodiments, R¹ is an alkenyl group (e.g., a vinyl group (H₂C═CH—), anallyl group (H₂C═CH—CH₂—), a vinyl ether group (H₂C═CH—O—), a styrylgroup (H₂C═CH—(C₆H₄)—), and the like). In some embodiments, R¹ is analkynyl group (e.g., an ethynyl group (HC—C—), a propargyl group(HC—C—CH₂—), a propargyl ether group (HC—C—CH₂—O—), and the like). Insome embodiments, R¹ is an aldehyde group (e.g., H(C═O)—, a benzaldehydegroup (H(C═O)—(C₆H₄)—, and the like). In some embodiments, R¹ is aketone group (e.g., (C₁₋₆ alkyl)-(C═O)—). In some embodiments, R¹ is athiol group, for example, a thiol group of the formula HS—. In someembodiments, R¹ is a pentafluorophenyl group. In some embodiments, R¹ isa zwitterionic group (e.g., of the formula A-B—C—, wherein A is a centerof permanent positive charge or a center of permanent negative charge; Bis a divalent group comprising a C₁₋₁₂ alkylene group, a C₆₋₃₀ aryleneor heteroarylene group, or an alkylene oxide group; and C is a center ofpermanent positive charge or a center of permanent negative charge,provided that the zwitterionic group has an overall net charge of zero).In some embodiments, R¹ is a pyridyl disulfide group. In someembodiments, R¹ is a glutathione group. In some embodiments, R¹ is athiamine group. In some embodiments, R¹ is a poly(ethylene glycol)group.

In some embodiments, R¹ is of the formula

wherein in the above formulas, R⁵ is hydrogen or a C₁₋₆ alkyl group,R^(d) is hydrogen or a C₁₋₆ alkyl group, m is an integer from greaterthan 1 to 900; A is a center of permanent positive charge or a center ofpermanent negative charge; B is a divalent group comprising a C₁₋₁₂alkylene group, a C₆₋₃₀ arylene or heteroarylene group, or an alkyleneoxide group; C is a center of permanent positive charge or a center ofpermanent negative charge, provided that the zwitterionic group has anoverall net charge of zero; and the curved line indicates the point ofattachment of the R¹ group to the rest of the temozolomide compound(e.g., via linking group L¹).

In some embodiments, when R¹ is a group of the formulaH₂C═C(R^(b))—(C═O)—O—, wherein R^(b) is methyl, hydrogen, fluoro, cyano,or trifluoromethyl; an aldehyde group; or a thiol group, X is —O—. Insome embodiments, when R¹ is an alkenyl group or an alkynyl group, X is—NH—.

In an embodiment, the temozolomide compound is of formula (I) and X is—O—; n is 1; L¹ is a divalent C₁₋₆ alkylene group; and R¹ is amethacrylate group. In some embodiments, L is a divalent C₂₋₆ alkylenegroup, preferably a C₂ alkylene group. For example, the temozolomidecompound can be of the formula

In an embodiment, the temozolomide compound is of formula (I) and X is—O—; n is 1; L¹ is a divalent C₁₋₆ alkylene group; and R¹ is an acrylategroup. In some embodiments, L is a divalent C₂₋₆ alkylene group,preferably a C₂ alkylene group. For example, the temozolomide compoundcan be of the formula

In an embodiment, the temozolomide compound is of formula (I) and X is—O—; n is 1; L¹ is a divalent C₁₋₆ alkylene group; and R¹ is amethacrylamide group. In some embodiments, L¹ is a divalent C₂₋₆alkylene group, preferably a C₂ alkylene group. For example, thetemozolomide compound can be of the formula

In an embodiment, the temozolomide compound is of formula (I) and X is—NH—; n is 1; L¹ is a divalent C₁₋₆ alkylene group; and R¹ is amethacrylamide group. In some embodiments, L¹ is a divalent C₂₋₆alkylene group, preferably a C₂ alkylene group. For example, thetemozolomide compound can be of the formula

In an embodiment, the temozolomide compound is of formula (I) and X is—NH—; n is 1; L¹ is a divalent C₁₋₆ alkylene group; and R¹ is anacrylamide group. In some embodiments, L¹ is a divalent C₂₋₆ alkylenegroup, preferably a C₂ alkylene group. For example, the temozolomidecompound can be of the formula

In an embodiment, the temozolomide compound is of formula (I) and X is—O—; n is 1; L¹ is a divalent di(C₁₋₆ alkylene) disulfide group; and R¹is a methacrylate group. In some embodiments, L¹ is a divalent di(C₂₋₆alkylene) disulfide group, preferably a divalent di(C₂ alkylene)disulfide group (e.g., —(C₂H₄)—S—S—(C₂H₄)—). For example, thetemozolomide compound can be of the formula

In an embodiment, the temozolomide compound is of formula (I) and X is—O—; n is 1; L¹ is a divalent C₁₋₆ alkylene group; and R¹ is an aldehydegroup or a ketone group. In some embodiments, L¹ is a divalent C₂₋₆alkylene group, preferably a C₂ alkylene group. In some embodiments,when R¹ is a ketone group, R⁵ can be a methyl group or an ethyl group,preferably a methyl group. For example, the temozolomide compound can beof the formula

In an embodiment, the temozolomide compound is of formula (I) and X is—O—; n is 1; L¹ is a divalent C₁₋₆ alkylene group; and R¹ is a thiolgroup. In some embodiments, L¹ is a divalent C₂₋₆ alkylene group,preferably a C₃ alkylene group. For example, the temozolomide compoundcan be of the formula

In an embodiment, the temozolomide compound is of formula (I) and X is—NH—; n is 1; L¹ is a divalent C₁_alkylene group; and R¹ is a vinylgroup. In some embodiments, L¹ is a divalent C₁₋₃ alkylene group,preferably a C₁ alkylene (e.g., a methylene) group. For example, thetemozolomide compound can be of the formula

In an embodiment, the temozolomide compound is of formula (I) and X is—NH—; n is 1; L¹ is a divalent C₁₋₆ alkylene group; and R¹ is an ethynylgroup. In some embodiments, L is a divalent C₁₋₃ alkylene group,preferably a C₁ alkylene (e.g., a methylene) group. For example, thetemozolomide compound can be of the formula

In an embodiment, the temozolomide compound is of formula (I) and X is—O—; n is 0; and R¹ is a pentafluorophenyl group. For example, thetemozolomide compound can be of the formula

In an embodiment, the temozolomide compound is of formula (I) and X is—O—; n is 1; L is a divalent C₁₋₆ alkylene group; and R¹ is a pyridyldisulfide group. In some embodiments, L¹ is a divalent C₁₋₃ alkylenegroup, preferably a C₂ alkylene (e.g., an ethylene) group. For example,the temozolomide compound can be of the formula

In an embodiment, the temozolomide compound is of formula (I) and X is—O— or —NH—; n is 1; L is a divalent C₁₋₁₂ alkylene sulfide group or adivalent C₁_alkylene group; and R¹ is a glutathione group. For example,the temozolomide compound can be of the formula

In an embodiment, the temozolomide compound is of formula (I) and X is—NH— or —O—; n is 1; L¹ is a divalent C₁₋₁₂ alkylene sulfide group; andR¹ is a thiamine group. For example, the temozolomide compound can be ofthe formula

In an embodiment, the temozolomide compound is of formula (I) and X is—O— or —NH—; n is 1; L¹ is a di(C₂₋₂alkylene) disulfide group or C₁₋₁₂alkylene sulfide group; and R¹ is a poly(ethylene glycol) group. Forexample, the temozolomide compound can be the formula

wherein m is an integer from greater than 1 to 900, or 1 to 500, or 1 to250, or 1 to 100, or 1 to 50.

In an embodiment, the temozolomide compound is of formula (I) and X is—O— or —NH—; n is 1; L¹ is a divalent di(C₂₋₁₂ alkylene) disulfidegroup, C₂₋₁₂ alkylene ester group, C₆₋₂₀ arylene group, C₁₋₂₀ alkyleneoxide group, or C₁₋₁₂ alkylene sulfide group; and R¹ is aphosphorylcholine zwitterionic group having the structure A-B—C—,wherein A is an ammonium group of the formula —N(R⁷)₃, wherein R⁷ is aC₁₋₆ alkyl group; B is a divalent C₁₋₆ alkylene group; and C is adivalent phosphate group. In another embodiment, X is —O— or —NH—; n is1; L is a divalent C₁₋₆ alkylene group; and R¹ is a sulfobetainezwitterionic group having the structure A-B—C-L²-, wherein A is asulfonate group; B is a divalent C₁₋₆ alkylene group; C is a divalentammonium group; and L² is a divalent C₁₋₆ alkylene group; wherein L² ofthe zwitterionic group is covalently bound to L¹ through a thioetherbond or a disulfide bond. In yet another embodiment, X is —O— or —NH—; nis 1; L¹ is a divalent C₁₋₆ alkylene group; and R¹ is a carboxybetainezwitterionic group having the structure A-B—C-L²-, wherein A is acarboxylate group; B is a divalent C₁₋₆ alkylene group; C is a divalentammonium group; and L² is a divalent C₁₋₆ alkylene group; wherein L² ofthe zwitterionic group is covalently bound to L through a thioether bondor a disulfide bond. For example, the temozolomide can be of the formula

In some embodiments, the R¹ group is preferably a functional group thatenables further functionalization of the temozolomide compound (i.e.,conjugation to a polymer scaffold or a water soluble group, as furtherdiscussed below, and as demonstrated in the working examples). Thus, R¹can preferably be a group of the formula H₂C═C(R^(b))—(C═O)—W—, whereinR^(b) is methyl, hydrogen, fluoro, cyano, or trifluoromethyl, and W is—O— or —NH—; an alkenyl group; an alkynyl group; an aldehyde group; aketone group; a thiol group, a pentafluorophenyl group, or a pyridyldisulfide group.

Thus, the above-described forms of the temozolomide compound describedherein can be particularly useful for conjugation (e.g., covalentattachment) of temozolomide to a drug delivery platform, for example, apolymer carrier. The polymer can be a homopolymer containing repeatingunits derived from the temozolomide compound, or can be a copolymercontaining repeating units derived from the temozolomide compound andother repeating units. The copolymer can be a random copolymer or ablock copolymer. The polymer carrier can be water soluble or waterinsoluble. The solubility of the polymer carrier can be selecteddepending on the desired method of administration of thetemozolomide-containing polymer to a subject (e.g., injectable deliveryvs. implantable delivery). In some embodiments, the polymer carrier ispreferably water soluble. As used herein, the term “water-soluble”refers to polymers that form a solution in water that is free ofinsoluble polymer particles. The determination that a solution is freeof insoluble polymer particles can be made using conventional lightscattering techniques or by passing the solution through a sufficientlyfine filter screen capable of capturing insoluble polymer particles. Forexample, a water soluble polymer can have a water solubility of at least10 milligrams per milliliter.

Thus, another aspect of the present disclosure is a polymer comprisingrepeating units comprising temozolomide, wherein the repeating units arederived from the above-described temozolomide compounds. In addition tothe repeating units comprising temozolomide derived from thetemozolomide compounds described above, the polymer can also optionallycomprise repeating units of formula (II), formula (III), or acombination thereof

wherein in each occurrence of the repeating units of formula (II), R² isindependently at each occurrence a hydrogen or a C₁₋₆ alkyl group, V isindependently at each occurrence —O— or —NH—, and R³ is independently ateach occurrence a zwitterionic group, a poly(C₁₋₆ alkylene oxide) group,a hydroxy-substituted C₁₋₆ alkyl group, or a C₁₋₁₂ alkyl group. In someembodiments, R² is preferably a C₁₋₆ alkyl group, more preferably amethyl group. In each occurrence of the repeating units of formula(III), R² is independently at each occurrence a hydrogen or a C₁₋₆ alkylgroup, and R⁶ is independently at each occurrence a substituted orunsubstituted C₆₋₂₀ aryl group, preferably wherein R² is hydrogen and R⁶is a C₆ aryl group (i.e., the repeating units according to formula (III)can be styrene repeating units).

In some embodiments, the polymer can be a homopolymer comprisingrepeating units comprising temozolomide, wherein the repeating units arederived from the above-described temozolomide compounds. The homopolymercan include 100 mole percent of repeating units comprising temozolomidebased on the total repeating units of the polymer. In some embodiments,the homopolymer can include less than 10 mole percent, or less than 5mole percent, or less than 1 mole percent of repeating units other thanrepeating units comprising temozolomide, based on the total repeatingunits of the polymer.

In some embodiments, the polymer can be a copolymer comprising repeatingunits comprising temozolomide and repeating units of formula (II)wherein R² is a C₁₋₆ alkyl group, preferably a methyl group, V is —O—,and R³ is a C₁₋₁₂ alkyl group, preferably a C₁₋₆ alkyl group, morepreferably a methyl group. In some embodiments, the polymer can be acopolymer comprising repeating units comprising temozolomide andrepeating units of formula (III) wherein R² is a hydrogen and R⁶ is asubstituted or unsubstituted C₆ aryl group. The copolymer can comprise 1to 95 mole percent, or 1 to 90 mole percent, or 1 to 75 mole percent, or1 to 60 mole percent, or 10 to 50 mole percent of repeating unitscomprising temozolomide based on the total repeating units of thecopolymer. The copolymer can be insoluble in water, for example, thecopolymer can have a solubility in water of less than 10 milligrams permilliliter, or less than 5 milligrams per milliliter, or less than 1milligram per milliliter.

In some embodiments, the polymer can be a copolymer comprising repeatingunits comprising temozolomide and repeating units of formula (II)wherein R² is a C₁₋₆ alkyl group, preferably a methyl group, V is —O—,and R³ is independently at each occurrence a zwitterionic group, apoly(C₁₋₆ alkylene oxide) group, or a hydroxy-substituted C₁₋₆ alkylgroup. The copolymer can comprise 1 to 95 mole percent, or 1 to 90 molepercent, or 1 to 75 mole percent, or 1 to 60 mole percent, or 5 to 55mole percent, or 10 to 55 mole percent, or 15 to 55 mole percent ofrepeating units comprising temozolomide. The copolymer can be watersoluble, wherein the term water soluble is as defined above.

In some embodiments, the polymer is a copolymer comprising repeatingunits of formulas (II) wherein R³ is a zwitterionic group. Azwitterionic group can have the structure -L²-A-B—C, wherein L² is adivalent linking group, A is a center of permanent positive charge or acenter of permanent negative charge, B is a divalent group comprising aC₁₋₁₂ alkylene group, a C₆₋₃₀ arylene group, or an alkylene oxide group,and C is a center of permanent negative charge or a center of permanentpositive charge, provided that the zwitterion has an overall net chargeof zero (i.e., the zwitterion is net neutral). For example, in anembodiment wherein A is a center of permanent positive charge, C is acenter of permanent negative charge. For example, in an embodimentwherein A is a center of permanent negative charge, C is a center ofpermanent positive charge. In some embodiments, a center of permanentpositive charge can include a quaternary ammonium group, a phosphoniumgroup, a sulfonium group, and the like. In some embodiments, the centerof permanent positive charge is preferably an ammonium group. In someembodiments, a center of permanent negative charge can include asulfonate group, a phosphonate group, a carboxylate group, a thiolategroup, and the like. The linking group L² is a divalent C₁₋₁₂ alkylenegroup, C₆₋₂₀ arylene group, or C₁₋₂₀ alkylene oxide group. In someembodiments, L² is preferably a divalent C₁₋₁₂ alkylene group, morepreferably a C₁₋₆ alkylene group.

In some embodiments, the zwitterionic group can be a phosphorylcholinegroup, a sulfobetaine group, or a carboxybetaine group. For example, thezwitterionic group can have the structure

wherein R^(c) is independently at each occurrence a substituted orunsubstituted C₁₋₁₂ alkyl group and p is independently at eachoccurrence an integer from 1 to 12 (e.g., p is 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, or 12). In some embodiments, each occurrence of R^(c) ismethyl. In some embodiments, p is an integer from 1 to 6, for example,in some embodiments p is equal to 2. In some embodiments, p is equal to3.

In some embodiments, R³ is preferably a phosphorylcholine zwitterionicgroup, for example a phosphorylcholine zwitterionic group of the formula

In some embodiments, R³ is a poly(C₁₋₆ alkylene oxide) group. Forexample, in some embodiments, R³ can be a poly(ethylene oxide) group, apoly(propylene oxide) group, and the like, or a combination thereof. Insome embodiments, R³ can be a poly(ethylene oxide) group. For example,the poly(ethylene oxide) group can be of the formula

wherein n is an integer from greater than 1 to 50, for example 2 to 50,or 5 to 25, R^(d) is a hydrogen or a C₁₋₆ alkyl group (e.g., a methylgroup), and the “*” indicates the point of attachment of the ethyleneoxide repeating unit to the polymer backbone (i.e., via V in formula(II)).

In some embodiments, R³ can be a hydroxy-substituted C₁₋₆ alkyl group.In some embodiments, the C₁₋₆ alkyl group can be an ethylene group, andR³ can be a 2-hydroxyethylene group. In some embodiments, the C₁₋₆ alkylgroup can be a propylene group, and R³ can be, for example, a2-hydroxypropylene group.

In some embodiments, the repeating units comprising temozolomide are offormula (IV)

wherein R⁴ is independently at each occurrence a hydrogen or a C₁₋₆alkyl group; Y is independently at each occurrence —O— or —NH—; L³ isindependently at each occurrence a divalent C₁₋₁₂ alkylene group,di(C₁₋₁₂ alkylene) disulfide group (e.g., —(C₁₋₁₂ alkylene)-S—S—(C₁₋₁₂alkylene)-), C₁₋₁₂ alkylene ester group (e.g., —(C₁₋₁₂alkylene)-(C═O)—O—), C₆₋₂₀ arylene group, or C₁₋₂₀ alkylene oxide group;Z is independently at each occurrence a disulfide group (e.g., —S—S—), athioether group (e.g., —S—(C₁₋₆ alkylene)-), a triazole group, ahydrazone group (e.g., —NH—N═CH—), or an amide group (e.g., —NH—(C═O)—);L¹ is independently at each occurrence a divalent C₁₋₁₂ alkylene group,di(C₁₋₁₂) alkylene disulfide group, C₁₋₁₂ alkylene ester group, C₆₋₂₀arylene group, C₁₋₂₀ alkylene oxide group, or C₁₋₁₂ alkylene sulfidegroup; X is independently at each occurrence —O— or —NH—; and p isindependently at each occurrence 0 or 1.

In an embodiment, the polymer is a copolymer comprising repeating unitsof formula (II) and formula (IV) wherein in each occurrence of therepeating units of formula (II), R² is a methyl group; V is —O—; and R³is a phosphorylcholine group having the structure

In each occurrence of the repeating units of formula (IV), R⁴ is amethyl group; Y is —O—; p is 0; L¹ is a divalent C₁₋₆ alkylene group(e.g., an ethylene group); and X is —O—. For example, the copolymer cancomprise repeating units of formula (IIA) and (IVA)

In an embodiment, the polymer is a copolymer comprising repeating unitsof formula (II) and formula (IV) wherein in each occurrence of therepeating units of formula (II), R² is a methyl group; V is —O—; and R³is a phosphorylcholine group having the structure

In each occurrence of the repeating units of formula (IV), R⁴ is amethyl group; Y is —O—; p is 0; L¹ is a divalent di(C₁₋₆ alkylene)disulfide group (e.g., a —(C₂H₄)—S—S—(C₂H₄)— group); and X is —O—. Forexample, the copolymer can comprise repeating units of formula (IIA) and(IVB)

In an embodiment, the polymer is a copolymer comprising repeating unitsof formula (II) and formula (IV) wherein in each occurrence of therepeating units of formula (II), R² is a methyl group; V is —O—; and R³is a phosphorylcholine group having the structure

In each occurrence of the repeating units of formula (IV), R⁴ is amethyl group; Y is —O—; p is 1; L³ is a divalent C₁₋₆ alkylene group; Zis a C₁-6 thioether group (e.g., —S—(C₁₋₆ alkylene)-); L¹ is a divalentC₁₋₆ alkylene group (e.g., a propylene group); and X is —NH—. Forexample, the copolymer can comprise repeating units of formula (IIA) and(IVC)

In an embodiment, the polymer is a copolymer comprising repeating unitsof formula (II) and formula (IV) wherein in each occurrence of therepeating units of formula (II), R² is a methyl group; V is —O—; and R³is a phosphorylcholine group having the structure

In each occurrence of the repeating units of formula (IV), R⁴ is amethyl group; Y is —O—; p is 1; L³ is a divalent C₁₋₂₀ alkylene oxidegroup; Z is a triazole group of the formula N N or

L¹ is a divalent C₁₋₆ alkylene group (e.g., a methylene group); and X is—NH—. For example, the copolymer can comprise repeating units of formula(IIA) and (IVD)

In an embodiment, the polymer is a copolymer comprising repeating unitsof formula (II) and formula (IV) wherein in each occurrence of therepeating units of formula (II), R² is a methyl group; V is —O—; and R³is a phosphorylcholine group having the structure

In each occurrence of the repeating units of formula (IV), R⁴ is amethyl group; Y is —O—; p is 1; L³ is a divalent C₁₋₆ alkylene group(e.g., a methylene group); Z is a hydrazone group (e.g.,—(C═O)—NH—N═CH—); L is a divalent C₁₋₆ alkylene group (e.g., an ethylenegroup); and X is —O—. For example, the copolymer can comprise repeatingunits of formula (IIA) and (IVE)

In an embodiment, the polymer is a copolymer comprising repeating unitsof formula (II) and formula (IV) wherein in each occurrence of therepeating units of formula (II), R² is a methyl group; V is —O—; and R³is a phosphorylcholine group having the structure

In each occurrence of the repeating units of formula (IV), R⁴ is amethyl group; Y is —O—; p is 0; and X is —NH—.

In an embodiment, the polymer is a copolymer comprising repeating unitsof formula (II) and formula (IV) wherein in each occurrence of therepeating units of formula (II), R² is a methyl group; V is —O—; and R³is a phosphorylcholine group having the structure

In each occurrence of the repeating units of formula (IV), R⁴ is amethyl group; Y is —O—; p is 1; L³ is a divalent C₁₋₁₂ alkylene group; Zis a disulfide group; L¹ is a divalent C₁₋₆ alkylene group; and X is—NH—.

In some embodiments, the copolymer can include 1 to 60 mole percent, or5 to 55 mole percent, or 10 to 55 mole percent, or 15 to 55 mole percentof repeating units comprising temozolomide (e.g., repeating unitsaccording to formula (IV)), based on the total moles of repeating unitsof the copolymer. The total amount of repeating units comprisingtemozolomide can be determined using known techniques, for example,using proton nuclear magnetic resonance (¹H NMR) spectroscopy or UV/Visspectroscopy.

In some embodiments, the copolymer can have a number average molecularweight of 1,000 to 100,000 grams per mole, or 5,000 to 100,000 grams permole, or 5,000 to 50,000 grams per mole, or 10,000 to 50,000 grams permole, or 10,000 to 40,000 grams per mole, or 10,000 to 30,000 grams permole, or 20,000 to 30,000 grams per mole. In some embodiments, thecopolymer can have a polydispersity index (PDI) of less than or equal to2, for example 1 to 2, or 1 to 1.75, or 1 to 1.5 or 1 to 1.2, or 1 to1.1. Molecular weight and PDI can be determined, for example, using gelpermeation chromatography (GPC), as described further in the workingexamples below.

In some embodiments, the copolymer can be a random copolymer. In someembodiments, the copolymer can be a block copolymer. In someembodiments, the copolymer is a diblock copolymer comprising repeatingunits according to formula (II) and (IV). In some embodiments, thecopolymer comprises less than or equal to 10 mole percent, or less than5 mole percent, or less than 1 mole percent of repeating units otherthan the repeating units according to formulas (II) and (IV).

In some embodiments, the copolymer is water soluble. For example, insome embodiments, the copolymer has a water solubility of at least 10milligrams per milliliter, or at least 25 milligrams per milliliter, orat least 50 milligrams per milliliter, or at least 100 milligrams permilliliter.

In some embodiments, the copolymer can be crosslinked. Crosslinking ofthe copolymer can be achieved, for example, by copolymerization with adifunctional monomer (e.g., a dimethacrylate monomer) or the like, or bya post-polymerization reaction using an appropriate crosslinker.Crosslinking of the copolymers can, in some embodiments, provide acrosslinked polymer gel, that is swellable, but not soluble, in anaqueous solution. In some embodiments, crosslinking of the copolymer canbe carried out such that crosslinked polymer nanoparticles comprisingthe copolymer are the result. Such polymer nanoparticles can have, forexample, an average diameter of less than or equal to 100 nanometers, or1 to 100 nanometers, or 10 to 100 nanometers, or 10 to 50 nanometers.Preferably, the polymer nanoparticles comprising the copolymer aredispersible in water.

The copolymers of the present disclosure can be prepared using methodswhich are generally known. For example, the copolymers can be preparedusing free radical polymerization techniques, for example controlledfree radical polymerization techniques (e.g., reversibleaddition-fragmentation chain-transfer polymerization (RAFT), and thelike). An example of the method of making the copolymer is furtherdescribed in the working examples below.

The structure of the repeating units comprising temozolomide will bedictated by the particular temozolomide derivative selected forpreparation of the copolymer. For example, the chemistry used to providethe repeating units comprising temozolomide (and thus the resultingrepeating unit structure) can be selected depending on the particular R¹group present on the temozolomide compound and, if necessary, thecomplementary reactive group present on the copolymer (e.g., ifpost-polymerization functionalization is used).

For example, in some embodiments, when a polymerizable group such as anacrylate, methacrylate, acrylamide, or methacrylamide is present on thetemozolomide compound, the copolymer can be prepared directly bycopolymerization of the temozolomide compound with the desired comonomerto provide the copolymer comprising repeating units of formula (II) and(IV).

In some embodiments, a copolymer precursor can be prepared, wherein theprecursor comprises repeating units of formula (II) and furthercomprises repeating units comprising a functional group that is reactivetowards the R¹ group of the temozolomide compound to be conjugated tothe precursor to provide the desired copolymer (i.e., the copolymercomprising repeating units of formula (II) and (IV)). For example, acopolymer precursor can comprise repeating units of formula (II) andrepeating units comprising an alkenyl group (e.g., when R¹ of thetemozolomide compound is a thiol, and the temozolomide compound can beconjugated to the precursor using a thiol-ene reaction), a thiol group(e.g., when the R¹ of the temozolomide compound is an alkenyl group or athiol group, and the temozolomide compound can be conjugated to theprecursor using a thiol-ene reaction or via disulfide formation,respectively), an azide group (e.g., when R¹ of the temozolomidecompound is an alkynyl group, and the temozolomide compound can beconjugated to the precursor using copper mediated azide-alkyne clickchemistry), or a hydrazide group (e.g., when R¹ of the temozolomidecompound is an aldehyde or ketone, and the temozolomide compound can beconjugated to the precursor via hydrazone formation).

The functionalized temozolomide compounds can also be conjugated topolymers not based on a (meth)acrylate polymer backbone. For example,the functional temozolomide compounds can also be conjugated to apolymer chain end, for example a poly(ethylene glycol chain end). Thus,another aspect of the present disclosure is a poly(ethyleneglycol)-temozolomide conjugate comprising a poly(ethylene glycol) havingat least two chain ends conjugated to a temozolomide compound.

The poly(ethylene glycol) can be a linear poly(ethylene glycol) (i.e.,having two chain ends) or a multi-arm branched or star poly(ethyleneglycol) (i.e., having more than 2 chain ends). In some embodiments, thepoly(ethylene glycol) having at least two chain ends conjugated to atemozolomide compound is of formula (V) or (VI)

wherein X is independently at each occurrence —O— or —NH—; L¹ isindependently at each occurrence a divalent C₁₋₁₂ alkylene group,di(C₁₋₁₂ alkylene) disulfide group, C₁₋₁₂ alkylene ester group, C₆₋₂₀arylene group, C₁₋₂₀ alkylene oxide group, or C₁₋₁₂ alkylene sulfidegroup; and q is an integer from 1 to 500, or 1 to 250, or 1 to 100, or 1to 50, or 1 to 25. In some embodiments, X is —O— and L¹ is a divalentdi(C₁₋₁₂ alkylene) disulfide group. In some embodiments, X is —NH— andL¹ is a divalent C₁₋₁₂ alkylene sulfide group.

As described above, the polymers described herein comprisingtemozolomide are thought to be particularly useful in the treatment ofcertain cancers. Thus, another aspect of the present disclosure is amethod of treating a disease. The method comprises administering atherapeutically effective amount of a composition comprising the abovedescribed polymer. The composition can include 1 to 100 weight percentof the polymer, based on the total weight of the composition. Thecomposition can be, for example, an aqueous composition comprising thecopolymer and an aqueous solution (e.g., water, a physiological salinesolution, a sugar solution, or combinations thereof). The compositioncan be administered to a subject having the disease to be treated. Thesubject can be a mammal, for example, a human, a mouse, or a rat. Insome embodiments, the disease to be treated is cancer, in particular,metastatic melanoma, high grade glioma, glioblastoma and other braincancers, lung cancer, breast cancer, testicular cancer, colon and rectalcancers, carcinomas, sarcomas, lymphomas, leukemias, and mycosisfungoides. In some embodiments, the disease is brain cancer, inparticular, high grade glioma, glioblastoma, and the like.Administration of the composition comprising the polymer can be, forexample, by injection, in particular intravenous injection, subcutaneousinjection, or intraperitoneal injection, oral administration, or byimplantation (e.g., of an implant device comprising the compositioncomprising the TMZ-containing polymer, preferably wherein theTMZ-containing polymer is not water-soluble). In some embodiments,administering the composition is preferably by intravenous injection.The therapeutically effective amount can be, for example, 75 to 200milligrams of TMZ per square meter of body surface area of the subject.Administration of the composition can be determined based on the needsof the subject, and can be of varying frequency, for example once perday, multiple (2 or more) times per day, once every three days, or anyother desired dosing frequency. The composition comprising the polymercan optionally further comprise one or more anti-cancer (e.g.,anti-brain cancer) ingredients or other additives to further enhance theefficacy of the composition or assist in manufacturing or storage of thecomposition, with the proviso that any additional anti-cancer ingredientor additive is selected so as to not have significant adverse effects onthe efficacy of the polymer comprising temozolomide.

The temozolomide compounds, polymers, and methods described herein arefurther illustrated by the following non-limiting examples.

Examples

The examples described herein utilize a novel methacrylate derivative ofTMZ, whereby TMZ was incorporated as pendent moieties in copolymers ofpoly(2-methacryloyloxyethyl phosphorylcholine) (polyMPC), awater-soluble biomaterial that has shown excellent utility for sustainedchemotherapy. See, e.g., Wong, K. E.; Mora, M. C.; Skinner, M.; McRaePage, S.; Crisi, G. M.; Arenas, R. B.; Schneider, S. S.; Emrick, T.Evaluation of PolyMPC-Dox Prodrugs in a Human Ovarian Tumor. Mol.Pharmaceutics 2016, 13, 1679-1687; McRae Page, S.; Henchey, E.; Chen,X.; Schneider, S.; Emrick, T. Efficacy of PolyMPC-DOX Prodrugs in 4T1Tumor-Bearing Mice. Mol. Pharmaceutics 2014, 11, 1715-1720.Copolymerization, achieved using reversible addition-fragmentationchain-transfer (RAFT) chemistry, afforded polyMPC-TMZ conjugates withtunable drug incorporations and narrow, well-defined molecular weightdistributions.

FIG. 1 depicts the synthesis of TMZ methacrylate (shown as compound 2).Room temperature diazotization and subsequent hydrolysis of theexocyclic carbamoyl was accomplished using nitrous acid, generated insitu at 0° C. from water, concentrated sulfuric acid, and sodiumnitrite. Precipitation over ice afforded TMZ-carboxylic acid (shown ascompound 1) as a fine white solid in yields of up to 79%. Disappearanceof resonances corresponding to primary amide protons in the protonnuclear magnetic resonance (H-NMR) spectrum indicated complete carbamoylhydrolysis, and electron ionization high-resolution mass spectrometry(HRMS-EI) confirmed the expected structure of compound 1([M]⁺: 195.0395g/mol).

Esterification chemistry was used, as shown in FIG. 1, to provideTMZ-methacrylate (shown as compound 2) by the reaction of compound 1 and2-hydroxyethyl methacrylate (HEMA) in the presence of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) andcatalytic 4-dimethylaminopyridine (DMAP). Given the poor solubility ofcompound 1 in organic solvents, esterification was performed indichloromethane (DCM) suspensions. Impurities and excess reagents werereadily removed by silica gel chromatography, yielding pure compound 2,as confirmed by fast-atom bombardment HRMS (HRMS-FAB) ([M+H]⁺: 308.0989g/mol), in 54% yield. Spectroscopy further demonstrated integrity of theimidazotetrazine heterocycle. In the ¹H-NMR spectrum of compound 2,resonances corresponding to imidazole (peak e, δ=8.85 ppm) andmethyltriazene (peak f, δ=3.88 ppm) protons were observed, and UV-Visabsorption spectra of TMZ (λ_(max)=325 nm) and derivative 2 (λ_(max)=323nm) were found to be equivalent (as shown in FIG. 2).

Copolymerization of MPC and TMZ-methacrylate by RAFT was initiallyattempted in a mixture of methanol (MeOH) and dimethylsulfoxide (DMSO),a mixture that proved useful for homogeneity of the reaction mixture.Although monomer conversion was high (87%), as estimated by ¹H-NMRspectroscopy, characterization of the isolated copolymer by UV-Visspectroscopy showed a significant shift in absorption maximum(λ_(max)=287 nm), a spectral change indicative of TMZ decomposition.This observation prompted investigation of the stability of TMZ inorganic solvents, a property not previously reported to our knowledge.Solutions of TMZ (0.01 milligram per milliliter concentration) in 1:1MeOH:DMSO, DMSO, ACN, and 2,2,2-trifluoroethanol (TFE) were incubated atroom temperature, as well as elevated temperatures (e.g., 50° C. and 70°C.). Analysis by UV-Vis spectroscopy, shown in FIGS. 3-6, showed thatthe absorption spectrum of TMZ remained unchanged after 24 hourincubation in DMSO and ACN at all temperatures. In contrast, TMZ heatedat 50° C. or 70° C. in the MeOH:DMSO mixture fully degraded after only 1hour, with significant degradation observed even at room temperature.Importantly, TMZ was found to be stable in TFE, also a good solvent forpolyzwitterions, at temperatures up to 70° C. for greater than 24 hours.The marked difference in stability of TMZ observed between MeOH and TFEwas attributed to the poor nucleophilicity of the latter, whichprecludes rapid imidazotetrazine solvolysis.

Based on the solvent studies, TFE was selected as the solvent for RAFTpolymerization in the following examples. A schematic illustration ofthe RAFT polymerization of MPC and TMZ-methacrylate is shown in FIG. 7.

Copolymerizations of MPC and compound 2 were performed at 70° C. in TFE,as shown in FIG. 4, utilizing 4,4′-azobis(4-cyanovaleric acid) (ACVA)and 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid as radicalinitiator (I) and chain-transfer agent (CTA), respectively. Reagentstoichiometry was established to yield copolymers with approximatenumber-average molecular weights (M) of 20,000 grams per mole (g/mole),and monomer feed ratios were intended to target incorporations ofcompound 2 of 11 (Polymer 3A), 21 (Polymer 3B), 35 (Polymer 3C), and 50(Polymer 3D) mole percent. Monomer conversions of 88-94% were achievedwith polymerization times of 6 to 9 hours. After quenchingpolymerizations by exposure to air, the crude polymer products werepurified by repeated precipitation from TFE into excess THF, followed bycentrifugal dialysis against aqueous 0.1 molar (M) hydrochloric acid.Purified conjugates were lyophilized, giving Polymers 3A-3D as pinksolids in yields of 60-74%.

UV-Vis and NMR spectroscopy were performed to demonstrate successfulincorporation of pendent TMZ in copolymers 3A-3D. FIG. 8 shows UV-Visabsorption spectra of conjugates 3A-3D. Each copolymer shows anabsorption maximum at 323 nanometers (nm), with no spectral featuresindicative of TMZ degradation. In the ¹H-NMR spectrum of copolymer 3D,shown in FIG. 9, resonances corresponding to intact imidazole (region“j”, δ=8.34-8.64 ppm) and methyltriazene (peak “k”, δ=3.89 ppm) groupsare clearly observed. Additionally, carbon resonances at 140.88, 131.41,and 38.03 parts per million (ppm) are present in the 1³C-NMR spectrum of3D that correspond to urea, imidazole, and methyltriazene moieties,respectively, confirming TMZ fidelity. Each copolymer possesses anabsorption maximum at 323 nm, with a notable absence of any spectralfeatures that would otherwise suggest TMZ degradation.

Using ¹H-NMR spectroscopy, copolymer compositions were estimated bycomparing relative signal intensities of the imidazole andtrimethylammonium (region “g”, δ=2.80-3.30 ppm) groups. Additionally,incorporation of compound 2 was estimated by measuring UV-Vis absorptionat λ=323 nm for solutions of 3A-3D in TFE. The weight percent ofcompound 2 in the polyMPC-TMZ copolymers was then determined using acalibration curve. Estimated incorporations of compound 2 are summarizedin Table 1. In general, the copolymer compositions estimated using bothspectroscopic techniques were in reasonable agreement with thetheoretical values.

TABLE 1 Target Target Measured Measured incorpor- incorpor-incorporation incorporation ation of ation of of compound of compoundPoly- compound compound 2 by NMR 2 by UV/Vis mer 2 (mol %) 2 (wt %) (mol%)^(a) (wt %)^(b) 3A 11 11 16 8 3B 21 21 25 17 3C 35 36 37 27 3D 50 5252 38 ^(a)Molar incorporations estimated by ¹H-NMR (500 MHz)spectroscopy in TFE-d₃. ^(b)Mass incorporation estimated by UV-Visspectroscopy of TFE solutions at polymer concentrations of 0.05 mg/ml.

Molecular weight characterization of Polymers 3A-3D was performed by gelpermeation chromatography (GPC) eluting in TFE, and molecular weightdistributions were observed to be narrow and monomodal, as shown in FIG.10. Copolymers were prepared with low PDI values, estimated relative topoly(methyl methacrylate) calibration standards, and estimated M valueswere in reasonable agreement with those targeted. Additionally, GPCchromatograms of polyMPC-TMZ copolymers obtained using detection by UVabsorbance showed a notable absence of residual (i.e., unconjugated)compound 2, confirming conjugate purity, as shown in FIG. 11. Thismolecular weight characterization demonstrates that the RAFTpolymerization strategy is amenable to preparing well-definedpolymer-TMZ conjugates using compound 2 as a comonomer.

TABLE 2 Mn (theoretical) Mn (GPC) Polymer (g/mol) (g/mol) PDI 3A 20,16028,730 1.09 3B 19,050 27,920 1.10 3C 19,410 25,860 1.09 3D 19,880 21,8801.08

Testing of the cytotoxic effects of polyMPC-TMZ on glioblastoma (GBM)cell lines utilized U87MG (TMZ sensitive cell line) and T98G(TMZ-resistant cell line). PolyMPC-TMZ random and block copolymers weretested with each cell line, as shown in FIGS. 12 and 13, respectively.U87MG cells were plated on 96 well plates and cultured overnight in DMEMmedia containing 10% FBS at 37° C. in a CO₂ incubator. The cells weretreated with different doses of TMZ (0-10000 μM), polyMPC-TMZ-17%(random copolymer with 17 mole percent TMZ monomer) (0-10000 μM),polyMPC-TMZ-26% (random copolymer with 26% TMZ monomer) (0-10000 μM),polyMPC-TMZ-33% (random copolymer with 33% TMZ monomer) (0-10000 μM) orpolyMPC-TMZ-50% random copolymer with 50 mole percent TMZ monomer)(0-10000 μM) and cell viability was tested on Day 7 using theCellTiter-Glo luminescent cell viability assay purchased from Promega,Inc. PolyMPC itself was used as a control polymer. Dose response curveswere generated and the IC₅₀ value for TMZ was ˜192 μM while polyMPCitself exhibited no toxicity whatsoever. All of the polyMPC-TMZconjugates were efficacious, with a modified toxicity response as istypical of polymer prodrugs which mask the toxicity of the drug prior tomaking them available to the tumor. PolyMPC-TMZ conjugates containing26-50 mole percent TMZ had IC₅₀ values of approximately 2-3 times thatof TMZ itself (1282 μM, 424.8 μM, 494.6 μM, and 674.1 μM for polyMPC-TMZcontaining 17, 26, 33, and 50% TMZ, respectively, for random copolymers,and 1095 μM, 396.1 μM, and 517.3 μM for polyMPC-TMZ containing 16, 25,and 35% TMZ, respectively, for block copolymers). The higher IC₅₀ valuesof polyMPC-TMZ will allow for introduction of larger drug doses in invivo experiments.

The polyMPC-TMZ conjugates were found to aggregate into polymericnanoparticles in aqueous solution due to sequestration of thehydrophobic TMZ, forming a core-type structure, surrounded bywater-soluble polyMPC corona, as illustrated schematically in FIG. 14.Such aggregates are useful for further encapsulation of additionaltherapeutic agents, for example additional, non-conjugated TMZ,O⁶-benzylguanine, or doxorubicin. In this process, the drugs aresolubilized by mixing them with aqueous polyMPC-TMZ suspensions, leadingto solubilization of the drugs by loading into the TMZ-rich core ofpolyMPC-TMZ structures. The TMZ example serves as a facile method foramplifying TMZ loading, while the use of O⁶-benzylguanine is intended tosensitize TMZ-resistant glioblastoma cells. Doxorubicin, achemotherapeutic which has shown activity against glioblastoma in cellculture, can be loaded into the aggregates using the cytotoxicpolyMPC-TMZ amphiphiles for a dual therapeutic.

In an exemplary preparation of TMZ-loaded polyMPC-TMZ nanoparticles, anaqueous solution of a polyMPC-TMZ block copolymer with a concentrationof 5 mg/mL was added to a vial containing TMZ powder (3.5 mg). Thesuspension was subjected to vortexing for 1-2 minutes to afford ahomogenous suspension with no evidence of residual TMZ powder. Thesuspension was filtered through a 0.45 micron cellulose acetate filtere,and the TMZ-loaded NPs were analyzed by UV-Vis spectroscopy. Asignificant increase in the absorbance of the characteristic TMZ peak(λ=328-336 nm), along with a red-shift in the absorption maximum(λ_(polyMPC-TMZ)=328 nm→λ_(NPs)=336 nm), suggests TMZ encapsulation intothe polyMPC-TMZ core, as shown in FIG. 15.

Thus, TMZ, a first-line chemotherapeutic indicated for treatingglioblastoma, was successfully incorporated into a series of polyMPCconjugates by controlled RAFT copolymerization utilizing a novelTMZ-methacrylate derivative. TMZ was introduced as a pendent moiety in atunable fashion at drug loadings of greater than 50 mole percent, andconjugates were prepared with narrow and controlled molecular weightdistributions. This synthetic demonstration reveals a method forpreparing well-defined TMZ-containing polymer therapeutics using simpleand effective polymerizations that are metal-free. Moreover, owing tothe ubiquitous reactivity of the TMZ-methacrylate derivative,opportunities for introducing TMZ into a variety of biocompatiblepolymer backbones are now possible, thus providing a platform fordevelopment a range of TMZ polymer therapeutics suitable for local andsystemic chemotherapy. As extended and dose-dense TMZ regimens haveshown clinical benefits, novel therapeutic modalities that augment thein vivo characteristics of TMZ stand to significantly improve theefficacy of chemotherapy for treating glioblastoma. Furthermore,functionalization of polymer therapeutics with biorecognition moietieshold promise for providing a mechanism for blood-brain barrier crossingof TMZ-containing polymers, including presentation of small molecules,peptides, and antibodies that facilitate transcytosis across theblood-brain barrier.

Various small molecule water soluble TMZ derivatives are alsoachievable, where the solubilizing group was a zwitterionic group (e.g.,a phosphorylcholine group, a sulfobetaine group, or a carboxy betainegroup), a glutathione group, a thiamine group, or poly(ethylene glycol)(e.g., one arm, two arm, and four arm derivatives). The synthesis ofthese derivatives is further described below. A general procedure forthe preparation of water-soluble TMZ small molecule prodrugs withthioether linkages follows. TMZ prodrugs with covalent thioetherlinkages are prepared using this general strategy: TMZ-alkene (1 molarequivalent), the thiol-bearing species (e.g., phosphorylcholine thiol,carboxybetaine thiol, sulfobetaine thiol, glutathione, thiamine, orpoly(ethylene glycol) thiol) (1 molar equivalent), and a radicalphotoinitiator (e.g., 2,2-dimethoxy-2-phenylacetophenone (DMPA)) aredissolved in an appropriate solvent, such as 2,2,2-trifluoroethanol forzwitterionic thiols, 0.1 M HCl for glutathione and thiamine-thiol, andDCM for poly(ethylene glycol) thiols. The reaction mixture is irradiatedat room temperature under 365 nm light for 1 hour. The resulting smallmolecule prodrugs are purified by repeated precipitation into THF ordiethyl ether or by reverse-phase high-pressure liquid chromatography.Polymeric prodrugs are purified by dialysis against appropriate organicsolvents (e.g., DCM, THF, or 2,2,2-trifluoroethanol). Diagnosticspectroscopy of the depicted prodrugs confirming TMZ conjugation andretention of its labile chemical structure include anticipated ¹H-NMRresonances corresponding to methyltriazene (δ ˜3.88 ppm) and imidazole(δ ˜8.88 ppm), as well as UV-Vis absorption at λ=323-325 nm.

TMZ prodrugs bearing a redox-sensitive disulfide linkage are preparedusing the following general strategy: TMZ-pyridyl disulfide (1 molarequivalent) and the thiol-bearing molecule (1 molar equivalent) aredissolved in an appropriate solvent, such as 2,2,2-trifluoroethanol forzwitterionic thiols, 0.1 M HCl for glutathione and thiamine-thiol, andDCM for poly(ethylene glycol) thiols, containing a catalytic amount ofacid (e.g., acetic acid) and stirred at room temperature for 16-24hours. The resulting small molecule prodrugs are typically purified byrepeated precipitation into tetrahydrofuran or diethyl ether; smallmolecule conjugates can also be purified by reverse-phase high-pressureliquid chromatography. Polymeric prodrugs are purified by dialysisagainst appropriate organic solvents (e.g., DCM, THF, or2,2,2-trifluoroethanol). Spectroscopic characterization confirmssuccessful TMZ conjugation, including ¹H NMR resonances for themethyltriazene (6-3.88 ppm) and imidazole (6-8.88 ppm) groups, as wellas a UV-Vis absorption maximum at λ=323-325 nm which confirms thepresence of the intact heterocyclic structure.

Experimental details follow.

Materials. 2-Methacryloyloxyethyl phosphorylcholine (MPC),2-hydroxyethyl methacrylate (HEMA), anhydrous acetonitrile,4-(dimethylamino)pydridine (DMAP), N,N′-dicyclohexylcarbodiimide (DCC),sodium nitrite, dimethylsulfoxide (DMSO), sodium trifluoroacetate,4-cyano-4-(phenylcarbonothioylthio)pentanoic acid, and4,4′-azobis(4-cyanovaleric acid) (ACVA) were purchased fromSigma-Aldrich. Anhydrous diethyl ether, dichloromethane (DCM), ethylacetate, hexanes, methanol (MeOH), ACN, concentrated sulfuric acid,tetrahydrofuran (THF) and hydrochloric acid (HCl) were purchased fromFisher Chemical. 2,2,2-trifluoroethanol (TFE) was purchased from AlfaAesar. Temozolomide (TMZ) and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) waspurchased from TCI America. Deuterated solvents were purchased fromCambridge Isotope Laboratories, Inc. Unless otherwise noted, allchemicals were used as received without further purification. Inhibitorwas removed from MPC prior to polymerizations according to knownmethods.

Instrumentation. ¹H-NMR (500 MHz), ¹³C-NMR (125 MHz), and ³¹P-NMR (202MHz) spectra were collected using a Bruker Ascend 500 spectrometerequipped with a Prodigy cryoprobe. High-resolution mass spectra (HRMS)data were obtained using a JEOL-700 MStation spectrometer equipped withelectron impact (EI) and fast atom bombardment (FAB) sources. UV-Visabsorption spectra were recorded on PerkinElmer Lambda 25 and Shimadzu3600 spectrophotometers. Molecular weight and polydispersity (PDI) ofcopolymers 3A-3D were estimated by gel permeation chromatography (GPC)in TFE containing 0.02 M sodium trifluoroacetate against poly(methylmethacrylate) standards. GPC was operated at 1.0 ml/min and 40° C. withan Agilent 1260 isocratic pump, an autosampler, a PLgel guard column (50mm×7.8 mm), two PLgel mixed C columns (300 mm×7.8 mm×5 mm), one PLgelmixed D column (300 mm×7.8 mm×5 mm), an Agilent 1260 refractive indexdetector, and an Agilent 1200 UV detector.

Synthesis of TMZ-carboxylic acid (1). TMZ-carboxylic acid (shown ascompound 1 in FIG. 1) was prepared following a previously describedprocedure. See, e.g., Arrowsmith, J.; Jennings, S. A.; Langnel, D. A.F.; Wheelhouse, R. T.; Stevens, M. F. G. Antitumour Imidazotetrazines.Part 39. Synthesis of Bis(Imidazotetrazines) with Saturated SpaceGroups. J. Chem. Soc., Perkin Trans. 2000, 1, 4432-4438. In a roundbottom flask charged with a stir bar and fitted with an addition funnel,TMZ (2.29 grams, 11.8 millimoles) was dissolved in concentrated H₂SO₄(23.6 milliliters), and the resulting yellow solution was cooled at 0°C. under nitrogen. A solution of sodium nitrite (2.51 grams, 36.4millimoles) in water (23.6 milliliters) was then added drop-wise over 45minutes, noting the evolution of a brown gas during addition. Themixture was allowed to warm to room temperature and was stirred undernitrogen, protected from light. After stirring for 17 hours, thesolution was cooled at 0° C., and the reaction was quenched with ice(61.26 grams). Further stirring at 0° C. resulted in the precipitationof 1 as a fine white solid, which was isolated by vacuum filtration,washed with cold water, and dried under high vacuum to afford 1 in 75%yield. ¹H-NMR (500 MHz, DMSO-d₆, δ, ppm): 3.88 (s, 3H), 8.82 (s, 1H),13.33 (br, 1H). ¹³C-NMR (125 MHz, DMSO-d₆, δ, ppm): 36.32, 127.78,129.09, 136.48, 139.10, 161.85. HRMS-EI (m/z): [M]⁺ calculated forC₆H₅N₅O₃: 195.0392, found: 195.0395.

Synthesis of TMZ-methacrylate (2). In a round-bottom flask charged witha stir bar, compound 1 (592.5 mg, 3.05 mmol) was suspended in DCM (20mL). HEMA (353 μL, 2.91 mmol) and catalytic DMAP (36.0 mg, 0.29 mmol)were added to the suspension, followed by EDC (674 mg, 3.51 mmol); themixture became homogeneous and red. After stirring under nitrogen atroom temperature for 14 hours, the mixture was filtered, and thefiltrate was diluted with DCM (30 mL) and washed with aqueous 0.1 M HCl(5×50 mL). The organic layer was dried over Na₂SO₄(s) and concentratedby rotary evaporation. The resulting white solid was dried under highvacuum, protected from light, to yield compound 2 in 71% yield. 1H-NMR(500 MHz, DMSO-d₆, δ, ppm): 1.87 (s, 3H), 3.88 (s, 3H), 4.43 (t, J=5 Hz,2H), 4.62 (t, 2H), 5.68 (s, 1H), 6.04 (s, 1H), 8.85 (s, 1H). 13C (125MHz, DMSO-d₆, δ, ppm): 17.93, 36.40, 62.49, 62.51, 126.15, 126.20,129.41, 135.60, 136.87, 138.93, 160.29, 166.41. HRMS-FAB (m/z): [M+H]⁺calculated for C₁₂H₁₄N₅O₅: 308.0995, found: 308.0989.

General Procedure for the Synthesis of Copolymers 3A-3D by RAFT. MPC andcompound 2 were dissolved in TFE at a total monomer concentration of 1molar in a 20 milliliter vial charged with a stir bar. ACVA and4-cyano-4-(phenylcarbonothioylthio)pentanoic acid were added as radicalinitiator (I) and chain-transfer agent (CTA), respectively, targeting[monomer]₀:[CTA]₀:[I]₀=68:5:1. The reaction solution was purged withnitrogen at 0° C. for 15 minutes, and then was stirred at 70° C. toinitiate polymerization, gauging monomer conversion by ¹H-NMRspectroscopy. After achieving monomer conversion greater than 88%, thepolymerization was allowed to cool to room temperature and was quenchedby exposure to air. The crude reaction mixture was precipitated from TFEinto THF (repeated three times) to remove unreacted monomer 2, and thepolymer was isolated by centrifugation. The polymer pellet was dissolvedin aqueous 0.1 M hydrochloric acid (10 milliliters), added to a 10,000molecular weight cutoff (MWCO) centrifugal dialysis membrane, andcentrifuged (4000×g, 20 minutes, room temperature). Filtrate wasdiscarded, and centrifugal dialysis was repeated two more times.Concentrated polymer was dissolved in aqueous 0.1 molar hydrochloricacid (3 milliliters), and lyophilization afforded polymers 3A-3D as pinksolids. Incorporation of TMZ-methacrylate 2 was estimated by ¹H-NMRspectroscopy by comparing relative signal intensities at 8.46 ppm (C—Hin TMZ) and 2.80-3.30 ppm (N—(CH₃)₃ in MPC). Copolymer yields were60-74%. ¹H-NMR (500 MHz, TFE-d₃, δ, ppm): 0.08-1.09 (br, 3H), 1.09-2.15(br, 2H), 3.05 ppm (s, 9H), 3.54 (br, 2H), 3.88 (s, 3H), 4.06 (br, 4H),4.32 (br, 2H), 4.52 (br, 4H), 8.46 (s, 1H). ¹³C-NMR (125 MHz, TFE-d₃, δ,ppm): 18.32, 19.81, 38.03, 46.54, 47.17, 55.97, 64.24, 64.99, 65.91,66.68, 68.09, 128.59, 131.41, 138.82, 140.88, 162.72, 180.09. ³¹P-NMR(202 MHz, TFE-d₃, 6, ppm): −2.36.

Measurement of TMZ-Methacrylate Incorporation by UV-Vis Spectroscopy.TMZ-methacrylate 2 was dissolved in TFE at concentrations ranging from0.0005 mg/ml to 0.025 mg/ml, and a calibration curve was constructedfrom UV-Vis absorbance values at 323 nm. PolyMPC-TMZ copolymers 3A-3Dwere dissolved at a concentration of 0.05 mg/ml, and UV-Vis absorbancevalues were measured at 323 nm. Mass incorporation of compound 2 wasthen determined using the calibration curve.

Synthesis of hydroxyethyl pyridyl disulfide. To a round bottom flaskcontaining a solution of Aldrithiol™-2 (8.43 g, 38.4 mmol) and aceticacid (732 μL, 12.8 mmol) in methanol (0.3 M) was added mercaptoethanol(1.8 mL, 25.6 mmol). The mixture was stirred for 24 hours at roomtemperature, then filtered to remove the thione by-product andconcentrated by rotary evaporation. The crude product was purified bycolumn chromatography over silica gel, eluting with ethylacetate/hexanes (30/70→40/60→50/50→60/40), to yield2-(2-(pyridin-2-yl)diuslfanyl)ethanol as a yellow oil. ¹H NMR (MeOD, 500MHz): δ 2.96 (t, J=7.5 Hz, 2H), 3.79 (t, J=7.5 Hz, 2H), 7.22-7.25 (m,1H), 7.79-7.86 (m, 2H), 8.42 (d, J=6 Hz, 1H) ppm. ¹³C NMR (MeOD, 125MHz) δ 148.96, 137.96, 121.04, 120.12, 59.20, 41.22 ppm.

Synthesis of TMZ-substituted pyridyl disulfide. In a vial charged with astir bar, 2-hydroxyethyl 2-pyridyl disulfide (714.2 mg, 3.8 mmol),TMZ—COOH (815 mg, 4.20 mmol), and 4-dimethylaminopyridine (DMAP) (43.0mg, 0.35 mmol) were suspended in dichloromethane (DCM) (25 mL).1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (1.1 g, 5.7 mmol)was added and the suspension was stirred at room temperature. After 24hours, the mixture was concentrated by rotary evaporation, then purifiedby passing through silica gel, eluting with ethyl acetate:hexanes(50:50→60:40→70:30→80:20→90:10→100:0).

Product fractions were combined and concentrated by rotary evaporation.Vacuum drying afforded TMZ-PDS as a white solid in 58% yield. ¹H-NMR(500 MHz, DMSO-d₆, δ, ppm): 3.27 (t, J=6 Hz), 3.90 (s), 4.56 (t, J=6Hz), 7.22 (dd, J=10 Hz), 7.76 (td), 7.88 (d, J=5 Hz), 8.45 (d, 5 Hz),8.88 (s). ¹³C-NMR (125 MHz, DMSO-d₆, δ, ppm): 36.42, 36.94, 62.16,119.27, 121.21, 126.15, 129.43, 136.85, 137.80, 138.93, 149.52, 158.96,160.16.

Synthesis of N-allyl substituted TMZ. In a typical reaction, TMZ—COOH(1.05 molar equivalents), allylamine (1 molar equivalent), and DMAP (0.1molar equivalents) are suspended in DCM. EDC (1.5 molar equivalents) isadded and the suspension is stirred at room temperature. After 16-24hours, the mixture is concentrated by rotary evaporation and passedthrough a silica gel column eluting with a mixture of ethylacetate:hexanes. Allyl-TMZ is isolated by vacuum filtration as a whitesolid. ¹H-NMR (500 MHz, DMSO-d₆, δ, ppm): 3.86 (s), 3.92, 5.08 (d, 10-11Hz), 5.15 (d, 17 Hz), 5.89 (m), 8.64 (s), 8.85 (s).

Synthesis of TMZ-pentafluorophenyl ester. In a typical reaction,TMZ-COOH (1.2 molar equivalents), pentafluorophenol (1 molarequivalents), and DMAP (0.1 molar equivalents) are suspended in DCM. EDChydrochloride (1.5 molar equivalents) is added, and the suspension isstirred at room temperature. After 16-24 h, the reaction mixture isconcentrated by rotary evaporation and purified by column chromatographyover silica gel, eluting with a mixture of ethyl acetate/hexanes.

Synthesis of thiamine bromide. In a typical reaction, to a solution ofthiamine (1 molar equivalents) in DCM (0.2 M) is added phosphorustribromide (1 equivalents) at 0° C. This mixture is stirred at roomtemperature for 16-24 h, and the product isolated by precipitation intodiethyl ether or related organic solvents.

Synthesis of thiamine thiol. In a typical reaction, potassiumthioacetate (1 molar equivalents) is added to a solution of Thiamine-Br(1 molar equivalent) in DMF, and the solution is stirred at roomtemperature. The reaction mixture is purified by precipitation into anorganic solvent (e.g., diethyl ether) to yield Thiamine-SAc which isthen deprotected by the addition of a primary amine (e.g., butylamine,10 molar equivalents) in an appropriate organic solvent (e.g., DMF) atroom temperature. Purification by precipitation into an organic solvent(e.g., diethyl ether) yields Thiamine-SH.

Synthesis of phosphorylcholine thiol. PC-thiol is prepared following amodified literature procedure: 2-hydroxyethyl disulfide (1 molar equiv.)and triethylamine (2.5 molar equiv.) are dissolved in anhydroustetrahydrofuran (1.0 M) and added to a solution of2-chloro-1,3,2-dioxaphospholane-2-oxide (2.5 molar equiv.) at ˜20° C.The reaction mixture is stirred at room temperature for 2 h, thenfiltered under nitrogen atmosphere and concentrated by rotaryevaporation to give the bis-substituted phospholane. Successfulsubstitution and retention of the ring structure is confirmed by ³¹P NMRspectroscopy with a characteristic resonance at ˜17 ppm corresponding tothe phosphorus atom of the intact ring. The bis-substituted phospholaneis then combined with trimethylamine (TMA, 6 molar equiv.) in anhydrousacetonitrile (2.0 M) at 0° C. in a pressure vessel. The reaction mixtureis heated to 70° C. for 24 h, then cooled and filtered to give thePC-disulfide intermediate. ³¹P NMR spectroscopy confirmed successfulring-opening by TMA by a resonance at ˜0 ppm corresponding to thephosphate. The PC-disulfide is then reduced to the thiol by dissolutionin methanol and mixing with agarose beads containing immobilizedtris(2-carboxyethyl) phosphine. The beads are removed by filtration, andprecipitation into an organic solvent such as diethyl ether yields thedesired PC—SH.

Synthesis of sulfobetaine thiol and carboxybetaine thiol. SB-thiol andCB-thiol are prepared by nucleophilic ring opening reactions of1,3-propane sultone and 0-propiolactone, respectively, usingbis(2-dimethylaminoethyl) disulfide dihydrochloride (DMAEDS).1,3-Propane sultone (10 molar equivalents) is dissolved in anhydrousacetonitrile (1.0 M) with DMAEDS (1 molar equivalents) and triethylamine(10 molar equivalents). The reaction is heated to reflux for severalhours and the product obtained is washed with anhydrous acetonitrile toyield SB-disulfide. ¹H NMR spectroscopy confirms the desired product bythe resonance corresponding to the cationic dimethylamine protons ((3.2ppm), as well as by mass spectroscopy. The disulfide product is thendissolved in methanol and mixed with agarose beads containingimmobilized tris(2-carboxyethyl) phosphine to reduce the disulfide.Following the reduction, the beads are removed by filtration, andprecipitation of the mixture into an appropriate organic solvent, suchas diethyl ether, yields SB—SH. NMR spectroscopy confirms the reductionof the disulfide and mass spectroscopy characterizes the molecularweight of the desired product.

The compounds, polymers, and methods of the present disclosure includeat least the following embodiments, which are non-limiting.

Embodiment 1: A temozolomide compound of structure (I)

wherein X is —O— or —NR^(a)—, wherein Ra is hydrogen or a C₁₋₆ alkylgroup; L¹ is a divalent C₁₋₁₂ alkylene group, di(C₂₋₁₂ alkylene)disulfide group, C₂₋₁₂ alkylene ester group, C₆₋₂₀ arylene group, C₁₋₂₀alkylene oxide group, or C₁₋₁₂ alkylene sulfide group; n is 0 or 1; andR¹ is a group of the formula H₂C═C(R^(b))—(C═O)—W—, wherein R^(b) ismethyl, hydrogen, fluoro, cyano, or trifluoromethyl, and W is —O— or—NH—; an alkenyl group; an alkynyl group; an aldehyde group; a ketonegroup; a thiol group; a pentafluorophenyl group, a pyridyl disulfidegroup, a zwitterionic group, a glutathione group, a thiamine group, or apoly(ethylene glycol) group.

Embodiment 2: The temozolomide compound of embodiment 1, wherein R¹ isof the formula

wherein in the above formulas, R⁵ is hydrogen or a C₁₋₆ alkyl group;R^(d) is hydrogen or a C₁_6 alkyl group; m is an integer from greaterthan 1 to 900, or 1 to 500, or 1 to 250, or 1 to 100, or 1 to 50; A is acenter of permanent positive charge or a center of permanent negativecharge; B is a divalent group comprising a C₁₋₁₂ alkylene group, a C₆₋₃₀arylene or heteroarylene group, or an alkylene oxide group; and C is acenter of permanent positive charge or a center of permanent negativecharge, provided that the zwitterionic group has an overall net chargeof zero.

Embodiment 3: The temozolomide compound of embodiment 1, wherein X is—O— or —NH—; n is 1; L is a divalent C₁₋₆ alkylene group; and R¹ is amethacrylate group or an acrylate group or acrylamide group.

Embodiment 4: The temozolomide compound of embodiment 1, wherein X is—O— or —NH—; n is 1; L is a divalent di(C₁₋₆ alkylene) disulfide group;and R¹ is a methacrylate group.

Embodiment 5: The temozolomide compound of embodiment 1, wherein X is—O— or —NH—; n is 1; L is a divalent C₁₋₆ alkylene group; and R¹ is amethacrylamide group.

Embodiment 6: The temozolomide compound of embodiment 1, wherein X is—O—; n is 1; L is a divalent C₁₋₆ alkylene group; and R¹ is an aldehydegroup or a ketone group.

Embodiment 7: The temozolomide compound of embodiment 1, wherein X is—O—; n is 1; L¹ is a divalent C₁₋₆ alkylene group; and R¹ is a thiolgroup.

Embodiment 8: The temozolomide compound of embodiment 1, wherein X is—NH—; n is 1; L is a divalent C₁₋₆ alkylene group; and R¹ is a vinylgroup.

Embodiment 9: The temozolomide compound of embodiment 1, wherein X is—NH—; n is 1; L is a divalent C₁₋₆ alkylene group; and R¹ is an ethynylgroup.

Embodiment 10: The temozolomide compound of embodiment 1, wherein X is—O—; n is 0; and R¹ is a pentafluorophenyl group.

Embodiment 11: The temozolomide compound of embodiment 1, wherein X is—O—; n is 1; L¹ is a divalent C₁₋₆ alkylene group; and R¹ is a pyridyldisulfide group.

Embodiment 12: The temozolomide compound of embodiment 1, wherein X is—O—; n is 1; L¹ is a divalent C₁₋₁₂ alkylene sulfide group or a divalentC₁₋₆ alkylene group; and R¹ is a glutathione group.

Embodiment 13: The temozolomide compound of embodiment 1, wherein X is—NH—; n is 1; L¹ is a divalent C₁₋₆ alkylene group; and R¹ is a thiaminegroup.

Embodiment 14: The temozolomide compound of embodiment 1, wherein X is—O—; n is 1; L¹ is a di(C₂₋₁₂ alkylene) disulfide group or C₁₋₁₂alkylene sulfide group; and R¹ is a poly(ethylene glycol) group.

Embodiment 15: The temozolomide compound of embodiment 1, wherein X is—O— or —NH—; n is 1; L¹ is a divalent di(C₂₋₁₂ alkylene) disulfidegroup, C₂₋₁₂ alkylene ester group, C₆₋₂₀ arylene group, C₁₋₂₀ alkyleneoxide group, or C₁₋₁₂ alkylene sulfide group; and R¹ is aphosphorylcholine zwitterionic group having the structure A-B—C—,wherein A is an ammonium group of the formula —N(R⁷)₃, wherein R⁷ is aC₁₋₆ alkyl group; B is a divalent C₁₋₆ alkylene group; and C is adivalent phosphate group.

Embodiment 16: The temozolomide compound of embodiment 1, wherein X is—O— or —NH—; n is 1; L¹ is a divalent C₁₋₆ alkylene group; and R¹ is asulfobetaine zwitterionic group having the structure A-B—C-L²-, whereinA is a sulfonate group; B is a divalent C₁₋₆ alkylene group; C is adivalent ammonium group; and L² is a divalent C₁₋₆ alkylene group;wherein L² of the zwitterionic group is covalently bound to L¹ through athioether bond or a disulfide bond.

Embodiment 17: The temozolomide compound of embodiment 1, wherein X is—O— or —NH—; n is 1; L¹ is a divalent C₁₋₆ alkylene group; and R¹ is acarboxybetaine zwitterionic group having the structure A-B—C-L²-,wherein A is a carboxylate group; B is a divalent C₁_6 alkylene group; Cis a divalent ammonium group; and L² is a divalent C₁₋₆ alkylene group;wherein L² of the zwitterionic group is covalently bound to L¹ through athioether bond or a disulfide bond.

Embodiment 18: The temozolomide compound of embodiment 1, wherein R¹ isa group of the formula H₂C═C(R^(b))—(C═O)—W—, wherein R^(b) is methyl,hydrogen, fluoro, cyano, or trifluoromethyl, and W is —O— or —NH—; analkenyl group; an alkynyl group; an aldehyde group; a ketone group; athiol group, a pentafluorophenyl group, or a pyridyl disulfide group.

Embodiment 19: A polymer comprising repeating units comprisingtemozolomide derived from the temozolomide compound of embodiment 18;and optionally, repeating units of formula (II), formula (III), or acombination thereof

wherein in each occurrence of the repeating units of formula (II) R² isa hydrogen or a C₁₋₆ alkyl group; V is —O— or —NH—; and R³ is azwitterionic group, a poly(C₁₋₆ alkylene oxide) group, ahydroxy-substituted C₁₋₆ alkyl group, or a C₁₋₁₂ alkyl group; andwherein in each occurrence of the repeating units of formula (III) R² isa hydrogen or a C₁₋₆ alkyl group; and R⁶ is a C₆₋₂₀ aryl group.

Embodiment 20: The polymer of embodiment 19, wherein the polymer is acopolymer comprising repeating units of formula (II), and wherein R³ isa zwitterion having the structure -L²-A-B—C; wherein L² is a divalentC₁₋₁₂ alkylene group, C₆₋₂₀ arylene group, or C₁₋₂₀ alkylene oxidegroup; A is a center of permanent positive charge or a center ofpermanent negative charge; B is a divalent group comprising a C₁₋₁₂alkylene group, a C₆₋₃₀ arylene or heteroarylene group, or a C₁₋₂₀alkylene oxide group; and C is a center of permanent positive charge ora center of permanent negative charge, provided that the zwitterion hasan overall net charge of zero.

Embodiment 21: The polymer of embodiment 19 or 20, wherein the polymeris a copolymer comprising repeating units of formula (II), and whereinR³ is a zwitterionic group of the formula

Embodiment 22: The polymer of any one of embodiments 19 to 21, whereinthe repeating units comprising temozolomide are of formula (IV)

wherein R⁴ is independently at each occurrence a hydrogen or a C₁₋₆alkyl group; Y is independently at each occurrence —O— or —NH—; L³ isindependently at each occurrence a divalent C₁₋₁₂ alkylene group,di(C₁₋₁₂ alkylene) disulfide group, C₁₋₁₂ alkylene ester group, C₆₋₂₀arylene group, or C₁₋₂₀ alkylene oxide group; Z is independently at eachoccurrence a disulfide group, a thioether group, a triazole group, ahydrazone group, or an amide group; L¹ is independently at eachoccurrence a divalent C₁₋₁₂ alkylene group, di(C₁₋₁₂) alkylene disulfidegroup, C₁₋₁₂ alkylene ester group, C₆₋₂₀ arylene group, C₁₋₂₀ alkyleneoxide group, or C₁₋₁₂ alkylene sulfide group; X is independently at eachoccurrence —O— or —NH—; and p is independently at each occurrence 0 or1.

Embodiment 23: The polymer of embodiment 22, wherein the polymer is acopolymer comprising repeating units of formula (II) and (IV), whereinin each occurrence of the repeating units of formula (II), R² is amethyl group; V is —O—; and R³ is a phosphorylcholine group having thestructure

and in each occurrence of the repeating units of formula (IV), R⁴ is amethyl group; Y is —O—; p is 0; L¹ is a divalent C₁₋₆ alkylene group;and X is —O—.

Embodiment 24: The polymer of embodiment 22, wherein the polymer is acopolymer comprising repeating units of formula (II) and (IV), whereinin each occurrence of the repeating units of formula (II), R² is amethyl group; V is —O—; and R² is a phosphorylcholine group having thestructure

and in each occurrence of the repeating units of formula (IV), R⁴ is amethyl group; Y is —O—; p is 0; L¹ is a divalent di(C₁₋₆ alkylene)disulfide group; and X is —O—.

Embodiment 25: The polymer of embodiment 22, wherein the polymer is acopolymer comprising repeating units of formula (II) and (IV), whereinin each occurrence of the repeating units of formula (II), R² is amethyl group; V is —O—; and R² is a phosphorylcholine group having thestructure

and in each occurrence of the repeating units of formula (IV), R⁴ is amethyl group; Y is —O—; p is 1; L³ is a divalent C₁_6 alkylene group; Zis a C₁_6 thioether group; L¹ is a divalent C₁₋₆ alkylene group; and Xis —NH—.

Embodiment 26: The polymer of embodiment 22, wherein the polymer is acopolymer comprising repeating units of formula (II) and (IV), whereinin each occurrence of the repeating units of formula (II), R² is amethyl group; V is —O—; and R² is a phosphorylcholine group having thestructure

and in each occurrence of the repeating units of formula (IV), R⁴ is amethyl group; Y is —O—; p is 1; L³ is a divalent C₁₋₂₀ alkylene oxidegroup; Z is a triazole group of the formula

L¹ is a divalent C₁₋₆ alkylene group; and X is —NH—.

Embodiment 27: The polymer of embodiment 22, wherein the polymer is acopolymer comprising repeating units of formula (II) and (IV), whereinin each occurrence of the repeating units of formula (II), R² is amethyl group; V is —O—; and R² is a phosphorylcholine group having thestructure

and in each occurrence of the repeating units of formula (IV), R⁴ is amethyl group; Y is —O—; p is 1; L³ is a divalent C₁₋₆ alkylene group; Zis a hydrazone group of the formula —(C═O)—NH—N═CH—; L is a divalentC₁₋₆ alkylene group; and X is —O—.

Embodiment 28: The polymer of embodiment 22, wherein the polymer is acopolymer comprising repeating units of formula (II) and (IV), whereinin each occurrence of the repeating units of formula (II), R² is amethyl group; V is —O—; and R² is a phosphorylcholine group having thestructure

and in each occurrence of the repeating units of formula (IV), R⁴ is amethyl group; Y is —O—; p is 0; L is a divalent C₁₋₆ alkylene group; andX is —NH—.

Embodiment 29: The polymer of embodiment 22, wherein the polymer is acopolymer comprising repeating units of formula (II) and (IV), whereinin each occurrence of the repeating units of formula (II), R² is amethyl group; V is —O—; and R² is a phosphorylcholine group having thestructure

and in each occurrence of the repeating units of formula (IV), R⁴ is amethyl group; Y is —O—; p is 1; L³ is a divalent C₁₋₁₂ alkylene group; Zis a disulfide group; L¹ is a divalent C₁₋₆ alkylene group; and X is—O—.

Embodiment 30: The polymer of any one of embodiments 19 to 29, whereinthe polymer comprises 1 to 100 mole percent of repeating unitscomprising temozolomide based on the total repeating units of thepolymer.

Embodiment 31: A poly(ethylene glycol)-temozolomide conjugate comprisinga poly(ethylene glycol) having at least two chain ends conjugated to atemozolomide compound.

Embodiment 32: The poly(ethylene glycol)-temozolomide conjugate ofembodiment 31, wherein the poly(ethylene glycol) having at least twochain ends conjugated to a temozolomide compound is of formula (V) or(VI)

wherein X is independently at each occurrence —O— or —NH—; L¹ isindependently at each occurrence a divalent C₁₋₁₂ alkylene group,di(C₁₋₁₂ alkylene) disulfide group, C₁₋₁₂ alkylene ester group, C₆₋₂₀arylene group, C₁₋₂₀ alkylene oxide group, or C₁₋₁₂ alkylene sulfidegroup; and q is an integer from 1 to 50.

Embodiment 33: A method of treating a disease, the method comprisingadministering a therapeutically effective amount of a compositioncomprising the polymer of any one of embodiments 19 to 29, thepoly(ethylene glycol)-temozolomide conjugate of embodiments 31 or 32, orthe temozolomide compound of any one of embodiments 12 to 17.

Embodiment 34: A polymer nanoparticle comprising a polymer of any one ofembodiments 20 to 29.

Embodiment 35: The polymer nanoparticle of embodiment 34, wherein thepolymer is a block copolymer or a random copolymer.

Embodiment 36: The polymer nanoparticle of embodiment 34 or 35, whereinthe polymer nanoparticle further comprises a therapeutic moietyencapsulated in the core of the nanoparticle.

Embodiment 37: The polymer nanoparticle of embodiment 36, therein thetherapeutic moiety comprises temozolomide, O⁶-benzylguanine,doxorubicin, or a combination comprising at least one of the foregoing.

Embodiment 38: A method of treating a disease, the method comprisingadministering a therapeutically effective amount of a compositioncomprising a plurality of polymer nanoparticles according to any one ormore of embodiments 34 to 37.

All cited patents, patent applications, and other references areincorporated herein by reference in their entirety. However, if a termin the present application contradicts or conflicts with a term in theincorporated reference, the term from the present application takesprecedence over the conflicting term from the incorporated reference.

All ranges disclosed herein are inclusive of the endpoints, and theendpoints are independently combinable with each other. Each rangedisclosed herein constitutes a disclosure of any point or sub-rangelying within the disclosed range.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. “Or” means “and/or” unless clearly stated otherwise. Further,it should further be noted that the terms “first,” “second,” and thelike herein do not denote any order, quantity, or importance, but ratherare used to distinguish one element from another. The modifier “about”used in connection with a quantity is inclusive of the stated value andhas the meaning dictated by the context (e.g., it includes the degree oferror associated with measurement of the particular quantity).

The term “alkyl” means a branched or straight chain, unsaturatedaliphatic hydrocarbon group, e.g., methyl, ethyl, n-propyl, i-propyl,n-butyl, s-butyl, t-butyl, n-pentyl, s-pentyl, and n- and s-hexyl.“Alkenyl” means a straight or branched chain, monovalent hydrocarbongroup having at least one carbon-carbon double bond (e.g., ethenyl(—HC═CH₂)). “Alkoxy” means an alkyl group that is linked via an oxygen(i.e., alkyl-O—), for example methoxy, ethoxy, and sec-butyloxy groups.“Alkylene” means a straight or branched chain, saturated, divalentaliphatic hydrocarbon group (e.g., methylene (—CH₂—) or, propylene(—(CH₂)₃—)). “Cycloalkylene” means a divalent cyclic alkylene group,—C_(n)H_(2n-x), wherein x is the number of hydrogens replaced bycyclization(s). “Cycloalkenyl” means a monovalent group having one ormore rings and one or more carbon-carbon double bonds in the ring,wherein all ring members are carbon (e.g., cyclopentyl and cyclohexyl).“Aryl” means an aromatic hydrocarbon group containing the specifiednumber of carbon atoms, such as phenyl, tropone, indanyl, or naphthyl.The prefix “halo” means a group or compound including one more of afluoro, chloro, bromo, or iodo substituent. A combination of differenthalo groups (e.g., bromo and fluoro), or only chloro groups can bepresent. The prefix “hetero” means that the compound or group includesat least one ring member that is a heteroatom (e.g., 1, 2, or 3heteroatom(s)), wherein the heteroatom(s) is each independently N, O, S,Si, or P. “Substituted” means that the compound or group is substitutedwith at least one (e.g., 1, 2, 3, or 4) substituents that can eachindependently be a C₁₋₉ alkoxy, a C₁₋₉ haloalkoxy, a nitro (—NO₂), acyano (—CN), a C₁₋₆ alkyl sulfonyl (—S(═O)₂-alkyl), a C₆₋₁₂ arylsulfonyl (—S(═O)₂-aryl) a thiol (—SH), a thiocyano (—SCN), a tosyl(CH₃C₆H₄SO₂—), a C₃₋₁₂ cycloalkyl, a C₂₋₁₂ alkenyl, a C₅₋₁₂cycloalkenyl, a C₆₋₁₂ aryl, a C₇₋₁₃ arylalkylene, a C₄₋₁₂heterocycloalkyl, and a C₃₋₁₂ heteroaryl instead of hydrogen, providedthat the substituted atom's normal valence is not exceeded. The numberof carbon atoms indicated in a group is exclusive of any substituents.For example —CH₂CH₂CN is a C₂ alkyl group substituted with a nitrile.

While particular embodiments have been described, alternatives,modifications, variations, improvements, and substantial equivalentsthat are or may be presently unforeseen may arise to applicants orothers skilled in the art. Accordingly, the appended claims as filed andas they may be amended are intended to embrace all such alternatives,modifications variations, improvements, and substantial equivalents.

1. A temozolomide compound of structure (I)

wherein X is —O— or —NR^(a)—, wherein Ra is hydrogen or a C₁₋₆ alkylgroup; L is a divalent C₁₋₁₂ alkylene group, di(C₂₋₁₂ alkylene)disulfide group, C₂₋₁₂ alkylene ester group, C₆₋₂₀ arylene group, C₁₋₂₀alkylene oxide group, or C₁₋₁₂ alkylene sulfide group; n is 0 or 1; andR¹ is a pentafluorophenyl group, a pyridyl disulfide group, azwitterionic group, a glutathione group, or a thiamine group; providedthat when R¹ is a pentafluorophenyl group, X is —O— and n is
 0. 2. Thetemozolomide compound of claim 1, wherein R¹ is of the formula

wherein in the above formulas, A is a center of permanent positivecharge or a center of permanent negative charge; B is a divalent groupcomprising a C₁₋₁₂ alkylene group, a C₆₋₃₀ arylene or heteroarylenegroup, or an alkylene oxide group; and C is a center of permanentpositive charge or a center of permanent negative charge, provided thatthe zwitterionic group has an overall net charge of zero.
 3. Thetemozolomide compound of claim 1, wherein X is —O—; n is 0; and R¹ is apentafluorophenyl group.
 4. The temozolomide compound of claim 1,wherein X is —O—; n is 1; L¹ is a divalent C₁₋₆ alkylene group; and R¹is a pyridyl disulfide group.
 5. The temozolomide compound of claim 1,wherein X is —O—; n is 1; L¹ is a divalent C₁₋₁₂ alkylene sulfide groupor a divalent C₁₋₆ alkylene group; and R¹ is a glutathione group.
 6. Thetemozolomide compound of claim 1, wherein X is —NH—; n is 1; L¹ is adivalent C₁₋₆ alkylene group; and R¹ is a thiamine group.
 7. Thetemozolomide compound of claim 1, wherein X is —O— or —NH—; n is 1; L¹is a divalent di(C₂₋₁₂ alkylene) disulfide group, C₂₋₁₂ alkylene estergroup, C₆₋₂₀ arylene group, C₁₋₂₀ alkylene oxide group, or C₁₋₁₂alkylene sulfide group; and R¹ is a phosphorylcholine zwitterionic grouphaving the structure A-B—C—, wherein A is an ammonium group of theformula —N(R⁷)₃, wherein R⁷ is a C₁₋₆ alkyl group; B is a divalent C₁₋₆alkylene group; and C is a divalent phosphate group.
 8. The temozolomidecompound of claim 1, wherein X is —O— or —NH—; n is 1; L¹ is a divalentC₁₋₆ alkylene group; and R¹ is a sulfobetaine zwitterionic group havingthe structure A-B—C-L²-, wherein A is a sulfonate group; B is a divalentC₁₋₆ alkylene group; C is a divalent ammonium group; and L² is adivalent C₁₋₆ alkylene group; wherein L² of the zwitterionic group iscovalently bound to L¹ through a thioether bond or a disulfide bond. 9.The temozolomide compound of claim 1, wherein X is —O— or —NH—; n is 1;L¹ is a divalent C₁₋₆ alkylene group; and R¹ is a carboxybetainezwitterionic group having the structure A-B—C-L²-, wherein A is acarboxylate group; B is a divalent C₁₋₆ alkylene group; C is a divalentammonium group; and L² is a divalent C₁₋₆ alkylene group; wherein L² ofthe zwitterionic group is covalently bound to L¹ through a thioetherbond or a disulfide bond.
 10. The temozolomide compound of claim 1,wherein R¹ is a pentafluorophenyl group or a pyridyl disulfide group.11. A polymer comprising repeating units comprising temozolomide derivedfrom the temozolomide compound of claim 10; and optionally, repeatingunits of formula (II), formula (III), or a combination thereof

wherein in each occurrence of the repeating units of formula (II) R² isa hydrogen or a C₁₋₆ alkyl group; V is —O— or —NH—; and R³ is azwitterionic group, a poly(C₁₋₆ alkylene oxide) group, ahydroxy-substituted C₁₋₆ alkyl group, or a C₁₋₁₂ alkyl group; andwherein in each occurrence of the repeating units of formula (III) R² isa hydrogen or a C₁₋₆ alkyl group; and R⁶ is a C₆₋₂₀ aryl group.
 12. Thepolymer of claim 11, wherein the polymer is a copolymer comprisingrepeating units of formula (II), and wherein R³ is a zwitterion havingthe structure -L²-A-B—C; wherein L² is a divalent C₁₋₁₂ alkylene group,C₆₋₂₀ arylene group, or C₁₋₂₀ alkylene oxide group; A is a center ofpermanent positive charge or a center of permanent negative charge; B isa divalent group comprising a C₁₋₁₂ alkylene group, a C₆₋₃₀ arylene orheteroarylene group, or a C₁₋₂₀ alkylene oxide group; and C is a centerof permanent positive charge or a center of permanent negative charge,provided that the zwitterion has an overall net charge of zero.
 13. Thepolymer of claim 11, wherein the polymer is a copolymer comprisingrepeating units of formula (II), and wherein R³ is a zwitterionic groupof the formula


14. The polymer of claim 11, wherein the repeating units comprisingtemozolomide are of formula (IV):

wherein R⁴ is independently at each occurrence a hydrogen or a C₁₋₆alkyl group; Y is independently at each occurrence —O— or —NH—; L³ isindependently at each occurrence a divalent C₁₋₁₂ alkylene group,di(C₁₋₁₂) alkylene disulfide group, C₁₋₁₂ alkylene ester group, C₆₋₂₀arylene group, or C₁₋₂₀ alkylene oxide group; Z is independently at eachoccurrence a disulfide group or an amide group; L¹ is independently ateach occurrence a divalent C₁₋₁₂ alkylene group, di(C₁₋₁₂ alkylene)disulfide group, C₁₋₁₂ alkylene ester group, C₆₋₂₀ arylene group, C₁₋₂₀alkylene oxide group, or C₁₋₁₂ alkylene sulfide group; X isindependently at each occurrence —O— or —NH—; and p is independently ateach occurrence 0 or
 1. 15. The polymer of claim 14, wherein the polymeris a copolymer comprising repeating units of formula (II) and (IV),wherein in each occurrence of the repeating units of formula (II), R² isa methyl group V is —O—; and R² is a phosphorylcholine group having thestructure

and in each occurrence of the repeating units of formula (IV), R⁴ is amethyl group; Y is —O—; p is 0; L¹ is a divalent di(C₁₋₆ alkylene)disulfide group; and X is —O—.
 16. The polymer of claim 14, wherein thepolymer is a copolymer comprising repeating units of formula (II) and(IV), wherein in each occurrence of the repeating units of formula (II),R² is a methyl group; V is —O—; and R² is a phosphorylcholine grouphaving the structure

and in each occurrence of the repeating units of formula (IV), R⁴ is amethyl group; Y is —O—; p is 1; L³ is a divalent C₁₋₁₂ alkylene group; Zis a disulfide group; L¹ is a divalent C₁₋₆ alkylene group; and X is—O—.
 17. The polymer of claim 14, wherein the polymer comprises 1 to 100mole percent of repeating units comprising temozolomide based on thetotal repeating units of the polymer.
 18. A method of treating adisease, the method comprising administering a therapeutically effectiveamount of a composition comprising the polymer of claim 14.